Optically controllable fgfr stimulation using wireless controlled cellular lighting system

ABSTRACT

The present invention relates to the field of stem cells. More specifically, the present invention provides compositions and methods for using optogenetics to sustain the pluripotency of stem cells. In one embodiment, a vector comprises a nucleotide sequencing encoding a fusion protein comprising the intracellular domain of fibroblast growth factor 1 receptor (FGFR1) and a photoactivatable domain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/962,246, filed Jan. 17, 2020, which is incorporated herein byreference in its entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under grant nos.NS093213 and AR070751 awarded by the National Institutes of Health, andgrant no. 1547515 awarded by the National Science Foundation. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of stem cells. Morespecifically, the present invention provides compositions and methodsfor using optogenetics to sustain the pluripotency of stem cells.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“P15986-02_ST25.txt.” The sequence listing is 26,756 bytes in size, andwas created on Jan. 19, 2021. It is hereby incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

Identification of channel rhodopsins as light-gated ion channels inalgal system [1], followed by various sensory photoreceptors, has openeda new chapter of optogenetics [2] in biological processes. Optogeneticsutilized key properties of sensory photoreceptors by sophisticatedmodulation of molecular targets with high spatial and temporal control,which certainly overcomes many drawbacks of conventional approaches.Current application of optogenetics is mostly limited to neurosciencefield relying on ion channels and light-driven ion pumps [3], but thereshould be plenty of other applications in many biology and medicine.

Previous pioneering studies have discovered several photoactivatableproteins, such as LOV (light-oxygen-voltage-sensing) domain [4,5],phytochrome B (PhyB) [6,7], and cryptochrome 2 (Cry2) [8]. Suchphotoactivatable proteins are key players for the optogenetic control ofintracellular signal transduction because, by absorbing energy from thephotons in excitation light, they can undergo conformational changes,rearrange inter- or intra-protein contacts, and modulate inter- orintra-protein interactions [9]. Such changes in photoactivable proteinscan mimic the dimerization of a signaling pathway, simply by lightillumination at a certain wavelength. For example, a photosensitiveprotein Cry2 homo-oligomerizes, and LOV domain homo-dimerizes, bothwithin seconds when illuminated with stimulatory light (˜470 nm) [9,10].Therefore, photoactivatable proteins are enticing light-sensing modulesfor use in optogenetic studies of intracellular signaling in mammaliansystems [11] because they have rapid responsiveness with high temporal(sub-second time resolution) and spatial (subcellular spatialresolution) precision, and they do not need exogenous cofactors orsignaling peptides.

Fibroblast growth factor 2 (FGF2) is a key component that promotesself-renewal and inhibits spontaneous differentiation of mamallianpluripotent stem cells (PSCs), including human PSC (hPSC) and porcineinduced PSC (piPSC) [12-19]. The human FGF family consists of 22 members(FGF1 to 23, except FGF15), which are structurally related signalingmolecules and subdivided into 7 subfamilies [20]. The mammalian FGFreceptor (FGFR) family, however, has only 4 highly conserved receptortyrosine kinases (FGFR1 to 4) [7]. Among them, FGFR1 predominantlyinteracts with FGF1 and FGF2 [21-23]. Like other receptor tyrosinekinases, FGFR1 dimerizes and trans-autophosphorylates a large number oftyrosine residues upon binding with FGF2, consequently activatingdownstream signaling pathways [24].

PSCs can be proliferated indefinitely and have the potential todifferentiate into many different cell types with promisingapplications. The establishment of a high-quality, high-precisionculture system is necessary for PSC applications; the existing cultureprotocols are completely dependent on recombinant proteins that arecritical culture medium components for the maintenance of PSCs[12-16,19]. However, high cost, thermal instability [25], and randomdistribution of the recombinant proteins impede large-scalemanufacturing. Here, we have developed a novel culture system for PSCsusing optical induction of the FGF signaling pathway. Our systemmaintained pluripotency of PSCs similar to the conventional culturesystem. Furthermore, the optically maintained PSCs displayed the abilityto differentiate into three germ layers, demonstrating that the opticalculture system can sustain the pluripotency of PSCs without exogeneousFGF2 protein supplementation.

SUMMARY OF THE INVENTION

The present invention provides one or more fusion protein embodimentshaving a cytoplasmic region of the FGF receptor-1 and alight-oxygen-voltage domain, which creates a tunable, bluelight-dependent activation of FGF signaling in human and porcinepluripotent stem cells (PSCs). The present invention also provides anovel PSC culture system which can include a wired or wirelesslycontrollable optical activation of the fibroblast growth factor (FGF)signaling pathway in the cells in culture without the need for dailysupplementation of recombinant FGF2 protein, a key molecule formaintaining pluripotency of PSCs. The present invention provides ahighly controllable optical stimulation of the FGF signaling pathwaysufficient for long-term maintenance of PSCs, without the loss ofdifferentiation potential into three germ layers. These embodimentsallow for culture systems that are a cost-effective platform for humanclinical trials and animal cellular agriculture

Therefore, in accordance with an embodiment, the present inventionprovides a synthetic polynucleotide comprising a sequence encoding theintracellular domain of the fibroblast growth factor 1 receptor (FGFR1)fused to a sequence encoding a photoactivatable light-oxygen-voltagesensing (LOV) domain.

In accordance with another embodiment, the present invention provides asynthetic polynucleotide comprising a sequence encoding theintracellular domain of the fibroblast growth factor 1 receptor (FGFR1)fused to a sequence encoding a photoactivatable light-oxygen-voltagesensing (LOV) domain comprising the nucleotide sequence of SEQ ID NO:6.

In accordance with an embodiment, the present invention provides asynthetic polynucleotide comprising a sequence encoding theintracellular domain of the fibroblast growth factor 1 receptor (FGFR1)fused to a sequence encoding a photoactivatable light-oxygen-voltagesensing (LOV) domain, a promoter sequence and a sequence encoding amyristolation signal peptide (Myr).

In accordance with an embodiment, the present invention provides anexpression vector comprising synthetic polynucleotide comprising asequence encoding the intracellular domain of the fibroblast growthfactor 1 receptor (FGFR1) fused to a sequence encoding aphotoactivatable light-oxygen-voltage sensing (LOV) domain.

In accordance with an embodiment, the present invention provides anexpression vector comprising synthetic polynucleotide comprising asequence encoding the intracellular domain of the fibroblast growthfactor 1 receptor (FGFR1) fused to a sequence encoding aphotoactivatable light-oxygen-voltage sensing (LOV) domain.

In accordance with another embodiment, the present invention provides aan expression vector comprising synthetic polynucleotide comprising asequence encoding the intracellular domain of the fibroblast growthfactor 1 receptor (FGFR1) fused to a sequence encoding aphotoactivatable light-oxygen-voltage sensing (LOV) domain, a promotersequence and a sequence encoding a myristolation signal peptide (Myr).

In accordance with another embodiment, the present invention provides aan expression vector comprising synthetic polynucleotide comprising asequence encoding the intracellular domain of the fibroblast growthfactor 1 receptor (FGFR1) fused to a sequence encoding aphotoactivatable light-oxygen-voltage sensing (LOV) domain, a promotersequence and a sequence encoding a myristolation signal peptide (Myr)comprising the nucleotide sequence of SEQ ID NO:1.

In accordance with an embodiment, the present invention providesexpression vectors comprising the synthetic polynucleotides describedherein.

In accordance with an embodiment, the present invention provides atransformant transformed by the vectors described herein, including, forexample, mammalian cells, and specifically, pluripotent stem cells.

In accordance with an embodiment, the present invention provides afibroblast growth factor free pluripotent stem cell culture systemcomprising one or more transformed cells as described herein and anillumination source.

In accordance with an embodiment, the present invention provides amethod for maintaining a pluripotent stem cell line in culturecomprising: a) a plurality of pluripotent stem cells in a fibroblastgrowth factor free culture medium, wherein said are transformed with theexpression vectors described herein; b) said transformed cells aremaintained in the cell culture system as described herein; and c)periodically illuminating the cells of b) at a wavelength of about 470nm for a sufficient time and intensity such that the cells maintaintheir pluripotent capability.

Accordingly, in one aspect, the present invention provides a vector. Inone embodiment, a vector comprises a nucleotide sequencing encoding afusion protein comprising the intracellular domain of fibroblast growthfactor 1 receptor (FGFR1) and a photoactivatable domain. In anotherembodiment, the fusion protein further comprises a signal peptide. In aspecific embodiment, the signal peptide comprises a myristolation signalpeptide (Myr). In a more specific embodiment, the Myr comprises theamino acid sequence of SEQ ID NO:4.

In another specific embodiment, the intracellular domain of FGFR1comprises the amino acid sequence of SEQ ID NO:6. In particularembodiments, the photoactivatable domain comprises alight-oxygen-voltage sensing (LOV) domain. In a specific embodiment, theLOV domain comprises the amino acid sequence of SEQ ID NO:8. In yetanother embodiment, the vector comprises the nucleotide sequence of SEQID NO:1. In certain embodiments, the present invention provides apluripotent stem cell (PSC) comprising a vector described herein.

In another aspect, the present invention provides a modified pluripotentstem cell. In one embodiment, the present invention provides a PSC whosegenome comprises a nucleotide sequence encoding a fusion proteincomprising the intracellular domain of FGFR1 and a photoactivatabledomain. In another embodiment, the present invention provides a PSC thatexpresses a fusion protein comprising the intracellular domain offibroblast growth factor 1 receptor (FGFR1) and a photoactivatabledomain.

In another embodiment, the fusion protein further comprises a signalpeptide. In a specific embodiment, the signal peptide comprises amyristolation signal peptide (Myr). In a more specific embodiment, theMyr comprises the amino acid sequence of SEQ ID NO:4. In anotherspecific embodiment, the intracellular domain of FGFR1 comprises theamino acid sequence of SEQ ID NO:6. In particular embodiments, thephotoactivatable domain comprises a light-oxygen-voltage sensing (LOV)domain. In a specific embodiment, the LOV domain comprises the aminoacid sequence of SEQ ID NO:8.

In a further aspect, the present invention provides a fibroblast growthfactor 2 (FGF2) free PSC culture system. In one embodiment, the culturesystem comprises a PSC described herein and a cell culture illuminationsource capable of illuminating the PSC with a selected wavelength andintensity of light. In specific embodiments, the cell cultureillumination source comprises a cell culture substrate, an upper andlower enclosure, one or more illumination sources, one or more circuitboards, and a microcontroller.

In particular embodiments, the illumination source can vary thewavelength of the illumination. In a specific embodiment, theillumination source emits light at a wavelength of about 470 nm. Inother embodiments, the illumination source comprises one or more lightemitting diodes (LEDs). In a specific embodiment, the illuminationsource continuously illuminates the cells for a period of time betweenabout 1 minute and about 120 minutes. In another specific embodiment,the illumination source illuminates the cells in a time interval ofbetween about 30 minutes to about 4 hours. In certain embodiments, theillumination source illuminates the cells at an intensity of betweenabout 0.1 μW/mm2 to about 25 μW/mm2. In particular embodiments, themicrocontroller is controlled through a computer server via either awireless or wired computer client.

In a further aspect, the present invention provides methods formaintaining the pluripotency of PSCs without exogenous FGF2supplementation. In a specific embodiment, the method comprises the stepof illuminating a PSC described herein with a light having a wavelengthof about 470 nm.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1H. Establishment of a novel and efficient FGF2-free hPSCculture system. FIG. 1A, 1B: A schematic illustration of the FGFsignaling pathway by (FIG. 1A) FGF ligand-elicited activation or by(FIG. 1B) an opto-FGFR activation via blue light illumination. FIG. 1C:A schematic illustration of the AAVS1 locus targeting using homologousrecombination enhanced by the CRISPR/Cas9 system. P1-P4 indicate the PCRprimer locations. Myr, myristoylation signal peptide; cytoFGFR1, thecytoplasmic region of FGFR1. FIG. 1D: A schematic illustration ofOpto-FGFR PSC maintenance (+F, 10 ng/mL FGF2 protein treatment, daily;+L, 1 μW/mm² at 470 nm for 5 min. every 2 h). FIG. 1E: Representativecolony morphologies of Opto-FGFR hESCs maintained in either +L conditionfor 54 weeks (left) or dark for 2 weeks (right). FIG. 1F, 1G: qRT-PCRresults of Opto-FGFR hPSCs cultured for 3 weeks (FIG. 1F, hESCs) or 12weeks (FIG. 1G, iPSCs) in either +F or +L condition (n=4; ns, notsignificant; unpaired t-test). All error bars represent mean±s.e.m. FIG.1H: Representative images of Opto-FGFR hiPSCs maintained in +L conditionfor 12 weeks showed undifferentiated colony morphology (left) andexpression of pluripotency markers TRA-1-81 (middle) and NANOG (right).Scale bars, 100 μm.

FIG. 2A-2F. The maintenance of pluripotency of Opto-FGFR hESCs culturedwith blue light illumination. FIG. 2A: A schematic illustration ofOpto-FGFR hESCs maintenance (+F, 10 ng/mL FGF2 protein treatmentsupplied daily; +L, 1 μW/mm² at 470 nm for 1-10 min. every 2 h; Diff.Ctrl, hESC-derived myotubes). FIG. 2B, 2C: Heat maps showing expressionlevels, (FIG. 2B) in log 2(FPKM) or (FIG. 2C) in FPKM, of specific genesin Opto-FGFR hESCs cultured in either +F, +L, or Diff. Ctrl conditionfor 3 weeks. FPKM, fragments per kilobase of transcript per millionmapped reads. FIG. 2D: A weighted Venn diagram between +F and +L groups,showing overlap of significantly differentially expressed genes comparedto the Diff. Ctrl group. FIG. 2E, 2F: Gene ontology (GO) analyses of(FIG. 2E) significantly upregulated genes and (FIG. 2F) downregulatedgenes in the +L group compared to the Diff. Ctrl group.

FIG. 3A-3G. In vitro and in vivo differentiation abilities of Opto-FGFRhPSCs maintained with blue light illumination into three germ layers.FIG. 3A: A schematic illustration of the differentiation of opticallymaintained Opto-FGFR hPSCs (1 μW/mm² at 470 nm for 5 min. every 2 h)into three germ layers. FIG. 3B-3G: Germ layer differentiation ofOpto-FGFR (FIG. 3B-3E, hESCs; fig, hiPSCs) in which cells were culturedwith blue light illumination. FIG. 3B, 3F: qRT-PCR results of theOpto-FGFR hPSCs, which were cultured with blue light illumination for 6weeks and then differentiated into three germ layers (n=4). All errorbars represent mean±s.e.m. FIG. 3C: Representative images of theOpto-FGFR hESCs, which were optically maintained for 10 weeks and thendifferentiated into three germ layers and immunostained with theindicated antibodies. FIG. 3D: Representative images of the Opto-FGFRhESCs, which were cultured with blue light illumination for 13 weeks,differentiated into dopaminergic neurons (left) and skeletal muscles(right), and immunostained with the indicated antibodies. FIG. 3E, 3G:Representative images of Opto-FGFR hPSC-derived tissues of three germlayers in in vivo teratoma assay. Scale bars, 100 μm.

FIG. 4A-4G. Establishment of a novel and efficient FGF2-free piPSCculture system that maintains pluripotency. FIG. 4A; A schematicillustration of the pRosa26 locus targeting using homologousrecombination enhanced by the CRISPR/Cas9 system. cytoFGFR1, thecytoplasmic region of the FGFR1. FIG. 4B: A schematic illustration ofOpto-FGFR piPSCs maintenance (+F, 10 ng/mL FGF2 protein treatment,daily; +L, 1 μW/mm² at 470 nm for 5 min. every 2 h). FIG. 4C:Representative colony morphologies of Opto-FGFR piPSCs maintained ineither +L condition (left) or dark (right) for 3 weeks.

FIG. 4D: A representative image of the Opto-FGFR piPSCs immunostainedwith OCT4 antibody after 3 weeks maintenance in +L condition. FIG. 4E:qRT-PCR results of Opto-FGFR piPSCs cultured in either +F or +Lcondition for 1 week (n=4; **, p value <0.01; *, p value <0.05; n.s.,not significant; unpaired t-test). FIG. 4F: A schematic illustration ofthe differentiation of optically maintained Opto-FGFR piPSCs in +Lcondition into three germ layers. FIG. 4G: qRT-PCR results of theOpto-FGFR piPSCs, which were maintained in +L condition for 3 weeks andthen differentiated into three germ layers (n=6). All error barsrepresent mean±s.e.m. Scale bars, 100 μm.

FIG. 5A-5F. Simple and cost-efficient LED illumination system for cellculture. FIG. 5A: A blown-out diagram of the LED illuminating device,consisting of the base casing, the microcontroller board (Arduino Uno),the LED circuit boards, and the top casing, with a standard 6-welltissue culture plate. FIG. 5B: Wire diagram between Arduino Uno and LEDcircuit boards. FIG. 5C: A fully-assembled LED illuminating device for6-well plates. FIG. 5D: Schematic overview of the wireless optogeneticsystem. FIG. 5E: The server computer's user interface (UI) where userscan control the phases of illumination and LED intensity and viewcurrent status of LED illumination. FIG. 5F: The mobile client's UIwhere users can wirelessly control phases of illumination and LEDintensity and view current status of LED illumination.

FIG. 6A-6F. The generation of opto-FGFR knock-in hPSCs. FIG. 6A:Validation of opto-FGFR gene cassette knock-in in hPSCs. Genomic DNAfrom the parental hESCs, Opto-FGFR hESCs, and Opto-FGFR hiPSCs wasamplified using the indicated primer pairs (Endogenous, P1 and P2; LeftArm, P1 and P3; Right Arm, P4 and P2; primer pair locations are shown inFIG. 1 c ). FIG. 6B, 6C: Representative colony morphologies and NANOGexpression of opto-FGFR knock-in (FIG. 6B) hESCs and (FIG. 6C) hiPSCs.FIG. 6D: PSC culture plates for FGF ligand treatment (left) and bluelight illumination (right). FIGS. 6E, 6F: The phosphorylation kineticsof (FIG. 6E) ERK1/2 and (FIG. 6F) MEK in the Opto-FGFR hESCs treatedwith FGF2 protein (red circles, n=4) or blue light (blue squares, n=4).All error bars represent mean±s.e.m. Scale bars, 100 μm.

FIG. 7A-7B. Phosphorylation of ERK1/2 in Opto-FGFR hESCs by blue lightillumination. FIG. 7A, 7B: The effect of duration (a, 1 μW/mm² at 470 nmfor 0-10 min) and intensity (b, 0-1 μW/mm² at 470 nm for 5 min) ofillumination on ERK1/2 phosphorylation (n=4; **, p value <0.01; *, pvalue <0.05; n.s., not significant; unpaired t-test). All error barsrepresent mean±s.e.m.

FIG. 8A-8C. Profiles of protein phosphorylation upon activation of theFGF signaling pathway. FIG. 8A: The heatmap shows comparable levels ofphosphorylation in multiple protein substrates in the FGF signalingpathway activated by FGF2 protein treatment (FGF, 10 ng/mL for 30 min;n=4) and blue light illumination (Opto, 1 μW/mm² at 470 nm for 30 min;n=4). FIG. 8B: The relative value of phosphorylation in the blue lightillumination and FGF2 protein treated groups. FIG. 8C: The ratio ofphosphorylated versus non-phosphorylated proteins in the blue lightillumination and FGF2 protein treated groups. All error bars representmean±s.e.m.

FIG. 9A-9F. Optogenetic activation of FGF signaling is sufficient tomaintain pluripotency in hESCs. FIG. 9A: A schematic illustration ofOpto-FGFR hESCs maintenance (+F, 10 ng/mL FGF2 protein treatmentsupplied daily; +L, 1 μW/mm² at 470 nm for 5 min. every 2 h; Untreated,maintained without FGF2 protein or blue light). FIG. 9B: Quantificationof FACS analyses of SSEA4⁺ cells from Opto-FGFR hESCs maintained in +F,+L, and Untreated conditions for 1 week (passage 1) or 3 weeks (passage3) (n=3; ***, p value <0.001; ns, not significant; unpaired t-test). Thecolored lines indicate each mean value. FIG. 9C: Representative FACSplots of OCT4::EGFP+ cells from Opto-FGFR hESCs cultured in either +F or+L condition for 3 weeks. FIG. 9D, 9E: Representative images of theOpto-FGFR hESCs immunostained with the indicated antibodies after (FIG.9D) 6 weeks or (FIG. 9E) 54 weeks maintenance in +L condition. FIG. 9F:A karyotype of Opto-FGFR hESCs cultured with blue light for 4 weeksshowed no aneuploidy and no structural rearrangements. Scale bars, 100μm.

FIG. 10A-10B. The freezing and thawing of Opto-FGFR hESCs cultured withblue light illumination. FIG. 10A: A schematic illustration of thefreezing and thawing procedure of optically maintained hESCs (1 μW/mm²at 470 nm for 5 min. every 2 h). Opto-FGFR hESCs cultured with bluelight illumination for 2 passages (2 weeks) were frozen into stock vials(stored in LN2 tank for 2 weeks) then thawed and maintained for 3 weekswith blue light illumination. FIG. 10B: A representative morphology ofthe resulting Opto-FGFR hESC colony (left) and the expression of NANOG(right). Scale bars, 100 μm.

FIG. 11A-11B. Gene expression profiles of pluripotency markers inOpto-FGFR hESCs at a single-cell level. FIG. 11A: A schematicillustration of Opto-FGFR hESC maintenance (+F, 10 ng/mL FGF2 proteintreatment supplied daily; +L, 1 μW/mm² at 470 nm for 5 min. every 2 h)and their single-cell qRT-PCR. OCT4::EGFP+ single-cells were captured byFACS sorting into a 96-well plate for qRTPCR. FIG. 11B: qRT-PCR resultsof Opto-FGFR hESCs cultured in either +F or +L condition for 45 weeks(n=78 for +F, n=88 for +L; n.s., not significant; unpaired t-test). Forbox and whisker plots, the black line across the box represents themedian, the box represents interquartile range and the whiskersrepresent the minimum and maximum.

FIG. 12A-12C. Scatter and volcano plots of global transcriptome analyses(RNA-seq). FIG. 12A: A schematic illustration of Opto-FGFR hESCsmaintenance (+F, 10 ng/mL FGF2 protein treatment supplied daily; +L, 1μW/mm² at 470 nm for 1 min. every 2 h). FIG. 12B: Scatter plot of globaltranscriptome changes between +F and +L conditions. The loge expressionlevels of genes from RNA-seq data are plotted. FPKM, fragments perkilobase of transcript per million mapped reads. FIG. 12C: Volcano plotof global transcriptome changes between +F and +L conditions. The loge(fold change) expression levels (+F vs +L) and −log₁₀(p value) of genesfrom RNA-seq data are plotted.

FIG. 13A-13F. The generation of opto-FGFR knock-in piPSCs. FIG. 13A: Thenumber of transfected cells and colonies formed after puromycinselection in each trial.

FIG. 13B: Representative colony morphology of opto-FGFR knock-in piPSCs.FIG. 13C: Phosphorylation of pErk1/2 in Opto-FGFR piPSCs in response toblue light illumination (1 μW/mm² at 470 nm for 5 min). A.U., arbitraryunit. FIG. 13D: A schematic illustration of Opto-FGFR piPSCs maintenance(+L, 1 μW/mm² at 470 nm for 5 min. every 2 h). FIG. 13E: Arepresentative image of alkaline phosphatase staining ofundifferentiated piPSCs maintained with blue light illumination for 1week. FIG. 13F: A karyotype of Opto-FGFR piPSC cultured with blue lightfor 3 weeks showed no aneuploidy and no structural rearrangements. Scalebars, 100 μm.

FIG. 14A-14E. Optogenetic activation of FGF signaling is sufficient tomaintain pluripotency of hESCs even in a feeder-free culture system.FIG. 14A-14C: Representative images of optically maintained Opto-FGFRhESCs (1 μW/mm² at 470 nm for 5 min. every 2 h) without feeder cells for3 weeks showed (FIG. 14A) undifferentiated colony morphology andexpression of pluripotency markers (FIG. 14B) OCT4 and (FIG. 14C) NANOG.FIG. 14D, 14E: Representative images of optically maintained Opto-FGFRhESCs (1 μW/mm² at 470 nm for 5 min. every 2 h) without feeder cells for8 weeks showed (FIG. 14D) undifferentiated colony morphology and (FIG.14E) no aneuploidy and no structural rearrangements in their karyotype.Scale bars, 100 μm.

FIG. 15A-15C. Cry2 PHR-based opto-FGFR system. FIG. 15A: Phosphorylationof ERK1/2 in response to blue light illumination (3.4-34 μW/mm² at 470nm for 1 min. every 10 min. for 3 cycles) in human embryonic kidneycells (293T) transiently transfected with Cry2 PHR-based opto-FGFR.PD173074 (0.2 μM) was used as an FGF signaling inhibitor. FIG. 15B: Arepresentative image of hESCs transiently transfected with Cry2PHR-based opto-FGFR. FIG. 15C: Phosphorylation of ERK1/2 in response toblue light illumination (34 μW/mm² at 470 nm for 1 min. every 10 min.for 3 cycles) in hESCs transiently transfected with Cry2 PHR-basedopto-FGFR. A.U., arbitrary unit. Scale bars, 100 μm.

FIG. 16 illustrates a schematic diagram of a general embodiment of theillumination system 100 of the present invention. The illuminationsystem 100 is adapted to illuminate cells, an in particular, transformedPSCs with the vectors of the present invention, to stimulate the FGFpathway and maintain the cells as pluripotent. The illumination system100 comprises, in one embodiment, LED illumination sources 101 which areembedded in the upper enclosure 104 which is shaped to accommodate acell culture substrate 106 in which the transformed PSCs are maintained.In an embodiment, the LEDs are individually connected to a printedcircuit board 102 which is connected to a microcontroller 103 placed inthe lower enclosure 105 and can be fastened together with fasteners.

FIG. 17 illustrates a schematic diagram of an embodiment of the circuitboard 102 and microcontroller 103 controlling the illumination sources101 of the present illumination system.

FIG. 18 illustrates a schematic diagram of an embodiment of thefibroblast growth factor free pluripotent stem cell culture system 200.The stem cell culture system 200 comprises at least one illuminationdevice 100 which is connected to a computer server 201 which controlsthe illumination parameters of the illumination sources 101 of thedevice 100 via wireless or wired connection to either a mobile clientcomputer 202 or client computer 203 with additional control software.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to theparticular methods and components, etc., described herein, as these mayvary. It is also to be understood that the terminology used herein isused for the purpose of describing particular embodiments only, and isnot intended to limit the scope of the present invention. It must benoted that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include the plural reference unless the contextclearly dictates otherwise. Thus, for example, a reference to a“protein” is a reference to one or more proteins, and includesequivalents thereof known to those skilled in the art and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Specific methods, devices, andmaterials are described, although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention.

All publications cited herein are hereby incorporated by referenceincluding all journal articles, books, manuals, published patentapplications, and issued patents. In addition, the meaning of certainterms and phrases employed in the specification, examples, and appendedclaims are provided. The definitions are not meant to be limiting innature and serve to provide a clearer understanding of certain aspectsof the present invention.

The present inventors have now developed a novel culture system for PSCsusing optical induction of the FGF signaling pathway. The inventivefusion protein and illumination system maintained pluripotency of PSCssimilar to the conventional culture system. Furthermore, the opticallymaintained PSCs displayed the ability to differentiate into three germlayers, demonstrating that the optical culture system can sustain thepluripotency of PSCs without exogenous FGF2 protein supplementation.

Previous reports have demonstrated the optical activation of receptortyrosine kinases in immortalized cell lines and developing embryos as aninitial step in evolving such optical technologies. Although a studyusing the optical control of ERK phosphorylation reported a perturbationof patterning and morphogenesis in Drosophila embryos, other studies didnot report any biologically relevant phenotypes; therefore, translatingthese approaches for use with PSCs has remained challenging.

The present inventors have now extended the approach to human andporcine PSCs and provide clear evidence that the inventive opto-FGFRPSCs are sufficiently maintained by blue light illumination and withoutthe need for FGF2 protein supplementation. Since there is no need tosupplement FGF2 protein, the media does not need to be changed on adaily basis in the optical PSC culture system. In feeder-free culturesystems, very high concentrations of FGF2 (up to 100 ng/mL) are requiredfor promoting self-renewal and inhibiting spontaneous differentiation ofhPSCs.

Even in a feeder-free culture system, the inventors succeeded inmaintaining pluripotency of hESCs using the inventive illuminatingculture system (FIG. 14 ). Moreover, the optically maintained PSCsappeared to retain the normal differentiation potential of traditionalPSCs.

There are various kinds of photoactivatable proteins available foroptogenetic approaches. Using the photolyase homology region of the Cry2protein (Cry2PHR39) and LOV domain. In the present invention, thephotosensory LOV domain served as an initiator in the light-inducedintermolecular signal transduction cascade of the FGF signaling pathway(termed as opto-FGFR signaling) for maintaining the pluripotency ofmammalian PSCs, without FGF2 protein supplementation. Although theinventors showed that they could optically activate FGF signaling inhuman embryonic kidney cells (HEK293T) and hESCs transiently transfectedwith the Cry2 PHR-based opto-FGFR (FIG. 15 ), the LOV domain wasselected as a photodimerizable domain because of two reasons: the LOVdomain has a shorter length (LOV, 432 bp or 144 aa26 vs Cry2 PHR, 1,506bp or 502 aa35), which is more favorable for knock-in, and LOV domainhas a shorter dissociation time after light withdrawal (LOV t112=2.5 minvs Cry2 PHR t112=5-6 min), which is more advantageous for accurateadjustment of the opto-FGFR signaling in PSC, than others. It isimportant to select an appropriate photosensory domain as thelight-sensing actuator module for a given optogenetic modulation systemin PSCs.

While it is a widely accepted idea that FGF2 protein directly stimulatesPSCs for their self-renewal, there are other possible mechanisms ofaction of FGF2, such as direct and indirect interaction and associationwith the IGF pathway. However, until now, there has been no experimentaltool to assess this possibility. The present invention herein providesthe first direct evidence that the molecular mechanism underlying thedaily supplementation with recombinant FGF2 protein to maintain thepluripotency of PSC is solely through the activation of the FGFsignaling pathway.

The prior art stem cell culture systems are heavily dependent on randomdistribution of expensive and thermo-unstable recombinant proteins in adish, which always can jeopardize future mass production of human stemcells for a population-wide cell therapy. The present inventiveopto-FGFR PSCs enable optical activation without any exogenous FGF2recombinant proteins and offer spatiotemporally precise optical controlof stem cell behaviors. Beyond implementing a new technology in the stemcell field, the present experimental data will help lead to thedevelopment of a potential therapeutic cure against many human diseasesby modulating multiple signaling pathways other than FGF2 and maximizingthe potential of human PSCs.

The prior art animal agriculture and husbandry methods cause significantenvironmental issues such as greenhouse gas emissions, fresh waterconsumption, and arable land usage. As an alternative, cellularagriculture or lab-grown meats' have been introduced and social consentis growing due to ethical and environmentally friendly concepts.However, the production costs and use of animal-derived thermo-unstablerecombinant proteins during the cellular agriculture process arebarriers to entering the market for mass consumption. Based on theinventors' calculation, recombinant FGF2 protein for 1 billion of PSCculture costs $14,000 weekly. In addition, due to thermal andchemo-physical instabilities, FGF2 is recommended to be freshly addedinto the media and it requires additional labor and miscellaneous fees.In conclusion, the present inventive opto-FGFR system in livestock PSCscan be a realistic solution to decrease these production costs.

Therefore, in accordance with an embodiment, the present inventionprovides a synthetic polynucleotide comprising a sequence encoding theintracellular domain of the fibroblast growth factor 1 receptor (FGFR1)fused to a sequence encoding a photoactivatable light-oxygen-voltagesensing (LOV) domain.

In an embodiment the LOV domain has the following nucleotide sequence(SEQ ID NO:7):

CCTGACTACAGTCTCGTGAAGGCTCTGCAAATGGCACAACAGAATTTTGTCATTACAGACGCCTCCCTCCCAGACAACCCTATCGTCTACGCCAGTAGAGGGTTTCTGACACTGACAGGCTATTCTCTCGACCAGATCCTGGGCAGGAACTGCAGGTTTCTGCAAGGGCCAGAAACAGACCCAAGAGCTGTGGATAAGATCAGGAATGCCATCACCAAAGGCGTTGATACCAGTGTCTGTCTGCTGAATTATAGACAGGATGGCACAACCTTCTGGAATCTCTTCTTCGTGGCTGGACTCAGAGATTCTAAGGGCAATATTGTCAACTACGTCGGAGTGCAGTCAAAGGTGAGCGAAGATTATGCCAAGCTGCTGGTCAACGAGCAGAACATTGAGTACAAAGGTGTGCGCACCAGTAACATGCTGCGCAGAAAG.

In accordance with another embodiment, the present invention provides asynthetic polynucleotide comprising a sequence encoding theintracellular domain of the fibroblast growth factor 1 receptor (FGFR1)fused to a sequence encoding a photoactivatable light-oxygen-voltagesensing (LOV) domain comprising the nucleotide sequence of SEQ ID NO:6.

The term “chimeric” or “fusion” polypeptide or protein refers to acomposition comprising at least one polypeptide or peptide sequence ordomain that is chemically bound in a linear fashion with a secondpolypeptide or peptide domain. One embodiment of this invention is anisolated or recombinant nucleic acid molecule encoding a fusion proteincomprising at least two domains, wherein the first domain comprises apolypeptide encoding FGFR1 protein, and the second domain comprising apolypeptide encoding a LOV protein.

In some embodiments, the fusion protein is designed for human PSCs.

In some alternative embodiments, the fusion protein is designed forporcine induced PSCs.

In accordance with an embodiment, the present invention provides asynthetic polynucleotide comprising a sequence encoding theintracellular domain of the fibroblast growth factor 1 receptor (FGFR1)fused to a sequence encoding a photoactivatable light-oxygen-voltagesensing (LOV) domain, a promoter sequence and a sequence encoding amyristolation signal peptide (Myr).

In accordance with an embodiment, the present invention provides anexpression vector comprising synthetic polynucleotide comprising asequence encoding the intracellular domain of the fibroblast growthfactor 1 receptor (FGFR1) fused to a sequence encoding aphotoactivatable proteins, including cryptochrome 2 (CRY2), light,oxygen, and voltage (LOV) domains, phytochrome B (PhyB) andUV-resistance locus 8 (UVR8) and its derivatives.

In some embodiments, the expression vector can be a plasmid.

In accordance with an embodiment, the present invention provides anexpression vector comprising synthetic polynucleotide comprising asequence encoding the intracellular domain of the fibroblast growthfactor 1 receptor (FGFR1) fused to a sequence encoding aphotoactivatable light-oxygen-voltage sensing (LOV) domain comprisingthe nucleotide sequence of SEQ ID NO:6.

In accordance with another embodiment, the present invention provides asynthetic polynucleotide comprising a sequence encoding theintracellular domain of the fibroblast growth factor 1 receptor (FGFR1)fused to a sequence encoding a photoactivatable light-oxygen-voltagesensing (LOV) domain, a promoter sequence and a sequence encoding amyristolation signal peptide (Myr). In an embodiment, theAAV-Myr-opto-FGFR-LOV comprising the nucleotide sequence of SEQ ID NO:1:

tgctttctctgaccagcattctctcccctgggcctgtgccgctttctgtctgcagcttgtggcctgggtcacctctacggctggcccagatccttccctgccgcctccttcaggttccgtcttcctccactccctcttccccttgctctctgctgtgttgctgcccaaggatgctctttccggagcacttccttctcggcgctgcaccacgtgatgtcctctgagcggatcctccccgtgtctgggtcctctccgggcatctctcctccctcacccaaccccatgccgtcttcactcgctgggttcccttttccttctccttctggggcctgtgccatctctcgtttcttaggatggccttctccgacggatgtctcccttgcgtcccgcctccccttcttgtaggcctgcatcatcaccgtttttctggacaaccccaaagtaccccgtctccctggctttagccacctctccatcctcttgctttctttgcctggacaccccgttctcctgtggattcgggtcacctctcactcctttcatttgggcagctcccctaccccccttacctctctagtctgtgctagctcttccagccccctgtcatggcatcttccaggggtccgagagctcagctagtcttcttcctccaacccgggcccctatgtccacttcaggacagcatgtttgctgcctccagggatcctgtgtccccgagctgggaccaccttatattcccagggccggttaatgtggctctggttctgggtacttttatctgtcccctccaccccacagtggggcaagcttctgacctcttctcttcctcccacagggcctcgagagatctggcagcggaGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGCTCGAGATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCtgatctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggtctcgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacacaagctggctagcgtttaaacgggccctctagactcgagcggccgcggccaccATGGGGAGTAGCAAGAGCAAGCCTAAGGACCCCAGCCAGCGCCTCGACATGAAGAGCGGCACCAAGAAGAGCGACTTCCATAGCCAGATGGCTGTGCACAAGCTGGCCAAGAGCATCCCTCTGCGCAGACAGGTAACAGTGTCAGCTGACTCCAGTGCATCCATGAACTCTGGGGTTCTCCTGGTTCGGCCCTCACGGCTCTCCTCCAGCGGGACCCCCATGCTGGCTGGAGTCTCCGAATATGAGCTCCCTGAGGATCCCCGCTGGGAGCTGCCACGAGACAGACTGGTCTTAGGCAAACCACTTGGCGAGGGCTGCTTCGGGCAGGTGGTGTTGGCTGAGGCCATCGGGCTGGATAAGGACAAACCCAACCGTGTGACCAAAGTGGCCGTGAAGATGTTGAAGTCCGACGCAACGGAGAAGGACCTGTCGGATCTGATCTCGGAGATGGAGATGATGAAAATGATTGGGAAGCACAAGAATATCATCAACCTTCTGGGAGCGTGCACACAGGATGGTCCTCTTTATGTCATTGTGGAGTACGCCTCCAAAGGCAATCTCCGGGAGTATCTACAGGCCCGGAGGCCTCCTGGGCTGGAGTACTGCTATAACCCCAGCCACAACCCCGAGGAACAGCTGTCTTCCAAAGATCTGGTATCCTGTGCCTATCAGGTGGCTCGGGGCATGGAGTATCTTGCCTCTAAGAAGTGTATACACCGAGACCTGGCTGCTAGGAACGTCCTGGTGACCGAGGATAACGTAATGAAGATCGCAGACTTTGGCTTAGCTCGAGACATTCATCATATCGACTACTACAAGAAAACCACCAACGGCCGGCTGCCTGTGAAGTGGATGGCCCCTGAGGCGTTGTTTGACCGGATCTACACACACCAGAGCGATGTGTGGTCTTTTGGAGTGCTCTTGTGGGAGATCTTCACTCTGGGTGGCTCCCCATACCCCGGTGTGCCTGTGGAGGAACTTTTCAAGCTGCTGAAGGAGGGTCATCGAATGGACAAGCCCAGTAACTGTACCAATGAGCTGTACATGATGATGCGGGACTGCTGGCATGCAGTGCCCTCTCAGAGACCTACGTTCAAGCAGTTGGTGGAAGACCTGGACCGCATTGTGGCCTTGACCTCCAACCAGGAGTATCTGGACCTGTCCATACCGCTGGACCAGTACTCACCCAGCTTTCCCGACACACGGAGCTCCACCTGCTCCTCAGGGGAGGACTCTGTCTTCTCTCATGAGCCGTATACCTGGGAGCCCTGTCTGCCTCGACACCCCACCCAGCTTGCCAACAGTGGACTCAAACGGCGCGTCGAGACCGGTCCTGACTACAGTCTCGTGAAGGCTCTGCAAATGGCACAACAGAATTTTGTCATTACAGACGCCTCCCTCCCAGACAACCCTATCGTCTACGCCAGTAGAGGGTTTCTGACACTGACAGGCTATTCTCTCGACCAGATCCTGGGCAGGAACTGCAGGTTTCTGCAAGGGCCAGAAACAGACCCAAGAGCTGTGGATAAGATCAGGAATGCCATCACCAAAGGCGTTGATACCAGTGTCTGTCTGCTGAATTATAGACAGGATGGCACAACCTTCTGGAATCTCTTCTTCGTGGCTGGACTCAGAGATTCTAAGGGCAATATTGTCAACTACGTCGGAGTGCAGTCAAAGGTGAGCGAAGATTATGCCAAGCTGCTGGTCAACGAGCAGAACATTGAGTACAAAGGTGTGCGCACCAGTAACATGCTGCGCAGAAAGCCCGGTGGATCCGGAGTCGACTATCCGTACGACGTACCAGACTACGCACTCGACtaagaattccaccacactggactagtggatccgagctcggtaccaagcttaagactagggacaggattggtgacagaaaagccccatccttaggcctcctccttcctagtctcctgatattgggtctaacccccacctcctgttaggcagattccttatctggtgacacacccccatttcctggagccatctctctccttgccagaacctctaaggtttgcttacgatggagccagagaggatcctgggagggagagcttggcagggggtgggagggaagggggggatgcgtgacctgcccggttctcagtggccaccctgcgctaccctctcccagaacctgagctgctctgacgcggctgtctggtgcgtttcactgatcctggtgctgcagcttccttacacttcccaagaggagaagcagtttggaaaaacaaaatcagaataagttggtcctgagttctaactttggctcttcacctttctagtccccaatttatattgttcctccgtgcgtcagttttacctgtgagataaggccagtagccagccccgtcctggcagggctgtggtgaggaggggggtgtccgtgtggaaaactccctttgtgagaatggtgcgtcctaggtgttcaccaggtcgtggccgcctctactccctttctctttctccatccttctttccttaaagagtccccagtgctatctgggacatattcctccgcccagagcagggtcccgcttccctaaggccctgctctgggcttctgggtttgagtccttggcaagcccaggagaggcgctcaggcttccctgtcccccttcctcgtccaccatctcatgcccctggctctcctgccccttccctacaggggttcctggctc tgctct.

In some embodiments, the plasmid comprises a AAV-CAGGS-eGFP vector wherethe eGFP region was replaced by a region of the opto-FGFR1 domain frommFGFR1-LOV vector to create a AAV-opto-FGFR.

It will be understood by those of ordinary skill in the art that in oneor more embodiments, one can knock-in the AAV-Myr-opto-FGFR-LOV plasmidinto the AAV1 locus (safe harbor area with constitutively expressedgene). It will also be understood that in other embodiments, the plasmidcan be incorporated into the genetically active loci of stem cells,including human stem cells.

As used herein, the term FGFRF1 means the protein encoded by the FGFR1gene, and which is a member of the fibroblast growth factor receptor(FGFR) family. The amino acid sequence is highly conserved betweenmembers and throughout evolution. FGFR family members differ from oneanother in their ligand affinities and tissue distribution. Afull-length representative protein would consist of an extracellularregion, composed of three immunoglobulin-like domains, a singlehydrophobic membrane-spanning segment and a cytoplasmic tyrosine kinasedomain. The extracellular portion of the protein interacts withfibroblast growth factors, setting in motion a cascade of downstreamsignals, ultimately influencing mitogenesis and differentiation. Amarked difference between this gene product and the other family membersis its lack of a cytoplasmic tyrosine kinase domain. The result is atransmembrane receptor that could interact with other family members andpotentially inhibit signaling. Multiple alternatively spliced transcriptvariants encoding the same isoform have been found for this gene

N-myristoylation is the attachment of a 14-carbon fatty acid, myristate,onto the N-terminal glycine residue of target proteins, catalyzed byN-myristoyltransferase (NMT), a ubiquitous and essential enzyme ineukaryotes. Many of the target proteins of NMT are crucial components ofsignalling pathways, and myristoylation typically promotes membranebinding that is essential for proper protein localization or biologicalfunction.

The term “variants” as used herein, means that the wild type amino acidsequences comprising the polypeptides of the compositions, may includesubstituted amino acids.

The term, “amino acid” includes the residues of the natural α-aminoacids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Lys, Ile, Leu,Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well asβ-amino acids, synthetic and unnatural amino acids. Many types of aminoacid residues are useful in the polypeptides and the invention is notlimited to natural, genetically-encoded amino acids. Examples of aminoacids that can be utilized in the peptides described herein can befound, for example, in Fasman, 1989, CRC Practical Handbook ofBiochemistry and Molecular Biology, CRC Press, Inc., and the referencecited therein. Another source of a wide array of amino acid residues isprovided by the website of RSP Amino Acids LLC.

The term “peptide” as used herein, includes a sequence of from four tosixteen amino acid residues in which the α-carboxyl group of one aminoacid is joined by an amide bond to the main chain (α- or β-)amino groupof the adjacent amino acid. The peptides provided herein for use in thedescribed and claimed methods and compositions can be cyclic.

In reference to the fusion polypeptide composition of the presentinvention, the functional portion can comprise, for instance, about 90%,95%, or more, of the FGFR1 and/or LOV polypeptide.

The functional portion of the fusion polypeptide composition of thepresent invention can comprise additional amino acids at the amino orcarboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of either of thewild type FGFR1 and/or LOV polypeptides. Desirably, the additional aminoacids do not interfere with the biological function of the functionalportion.

Included in the scope of the invention are functional variants of theinventive polypeptides, and proteins described herein. The term“functional variant” as used herein refers to either the FGFR1 and/orLOV polypeptide, or fusion protein having substantial or significantsequence identity or similarity to the FGFR1 and/or LOV polypeptide, orfusion protein, which functional variant retains the biological activityof the FGFR1 and/or LOV polypeptide, or fusion protein of which it is avariant. In reference to the original FGFR1 and/or LOV polypeptide, orprotein, the functional variant can, for instance, be at least about30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence tothe FGFR1 and/or LOV polypeptide, or protein.

The functional variant can, for example, comprise the amino acidsequence of the FGFR1 and/or LOV polypeptide fusion protein with atleast one or more conservative amino acid substitutions. Conservativeamino acid substitutions are known in the art, and include amino acidsubstitutions in which one amino acid having certain physical and/orchemical properties is exchanged for another amino acid that has thesame chemical or physical properties. For instance, the conservativeamino acid substitution can be an acidic amino acid substituted foranother acidic amino acid (e.g., Asp or Glu), an amino acid with anonpolar side chain substituted for another amino acid with a nonpolarside chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val,etc.), a basic amino acid substituted for another basic amino acid (Lys,Arg, etc.), an amino acid with a polar side chain substituted foranother amino acid with a polar side chain (Asn, Cys, Gln, Ser, Thr,Tyr, etc.), etc.

Functional variants can also include extensions of the FGFR1 and/or LOVpolypeptide fusion protein. For example, a functional variant of theFGFR1 and/or LOV polypeptide fusion protein can include 1, 2, 3, 4 and 5additional amino acids from either the N-terminal or C-terminal end ofthe FGFR1 and/or LOV polypeptide fusion protein.

Alternatively or additionally, the functional variants can comprise theamino acid sequence of the FGFR1 and/or LOV polypeptide fusion proteinwith at least one non-conservative amino acid substitution. In thiscase, it is preferable for the non-conservative amino acid substitutionto not interfere with or inhibit the biological activity of thefunctional variant. Preferably, the non-conservative amino acidsubstitution enhances the biological activity of the functional variant,such that the biological activity of the functional variant is increasedas compared to the FGFR1 and/or LOV polypeptide fusion protein.

The FGFR1 and/or LOV polypeptide fusion protein can consist essentiallyof the specified amino acid sequence or sequences described herein, suchthat other components of the functional variant, e.g., other aminoacids, do not materially change the biological activity of thefunctional variant.

It will be understood by those of ordinary skill in the art that theorientation of the two proteins in the fusion protein construct can bereversed, i.e., the N-terminal protein can comprise the LOV protein andthe C-terminal protein can comprise the FGFR1 protein.

Analogs and mimetics include molecules which include molecules whichcontain non-naturally occurring amino acids or which do not containamino acids but nevertheless behave functionally the same as thepeptide. Natural product screening is one useful strategy foridentifying analogs and mimetics.

Examples of incorporating non-natural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,omithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine and/or D-isomers of amino acids. A partial list of knownnon-natural amino acid contemplated herein is shown in Table 1.

TABLE 1 Non-natural Amino Acids Non-conventional amino acid CodeNon-conventional amino acid Code α-aminobutyric acid AbuL-N-methylalanine Nmala α-amino-α-methylbutyrate MgabuL-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagineNmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methyleysteine Nmcys aminonorbomyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Deys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nruval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-y-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamyimethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cyclododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Druncys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DumpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nieu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Druntyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methyIpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Neys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methyleysteine MeysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methyllencine Mien L-α-methyllysine Miys L-α-methylmethionine MmetL-α-methylnorleucine Mule L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nruhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane

Analogs of the subject peptides contemplated herein includemodifications to side chains, incorporation of non-natural amino acidsand/or their derivatives during peptide synthesis and the use ofcrosslinkers and other methods which impose conformational constraintson the peptide molecule or their analogs.

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzenesulphonic acid (TNBS); acylation of amino groups with succinic anhydrideand tetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitization, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Crosslinkers can be used, for example, to stabilize 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety such as maleimido or dithiomoiety (SH) or carbodiimide (COOH). In addition, peptides can beconformationally constrained by, for example, incorporation of C_(α) andN_(α)-methylamino acids, introduction of double bonds between C_(α) andC_(β) atoms of amino acids and the formation of cyclic peptides oranalogues by introducing covalent bonds such as forming an amide bondbetween the N and C termini, between two side chains or between a sidechain and the N or C terminus.

In some embodiments, the FGFR1 protein in the fusion protein ismammalian. In certain embodiments, the FGFR1 protein can be murine,porcine, ovine, bovine, human, or combinations thereof.

In accordance with an embodiment, the present invention provides acomposition comprising a polypeptide encoding human FGFR1 protein, or afunctional portion or fragment, or variant thereof, linked to apolypeptide encoding LOV protein, or a functional portion or fragment,or variant thereof.

In accordance with an embodiment, the present invention providesexpression vectors comprising the synthetic polynucleotides describedherein. In one embodiment, the present invention provides an expressioncassette specific for use with the CRISPR/Cas9 system. In an embodiment,a gRNA sequence (GTCCCCTCCACCCCACAGTG) (SEQ ID NO:9 is used to targetAAVS1 locus.

A “CRISPR,” “CRISPR system system,” or “CRISPR nuclease system” andtheir grammatical equivalents can include a non-coding RNA molecule(e.g., guide RNA) that binds to DNA and Cas proteins (e.g., Cas9) withnuclease functionality (e.g., two nuclease domains). See, e.g., Sander,J. D., et al., “CRISPR-Cas systems for editing, regulating and targetinggenomes,” Nature Biotechnology, 32:347-355 (2014); see also e.g., Hsu,P. D., et al., “Development and applications of CRISPR-Cas9 for genomeengineering,” Cell 157(6):1262-1278 (2014).

The term “gene editing” and its grammatical equivalents as used hereincan refer to genetic engineering in which one or more nucleotides areinserted, replaced, or removed from a genome. Gene editing can beperformed using a nuclease (e.g., a natural-existing nuclease or anartificially engineered nuclease).

The term “safe harbor” and “immune safe harbor”, and their grammaticalequivalents as used herein can refer to a location within a genome thatcan be used for integrating exogenous nucleic acids wherein theintegration does not cause any significant effect on the growth of thehost cell by the addition of the nucleic acid alone. Non-limitingexamples of safe harbors can include HPRT, AAVS SITE (e.g., AAVS1,AAVS2, etc.), CCR5, or Rosa26.

Site specific gene editing can be achieved using non-viral gene editingsuch as CRISPR, TALEN (see U.S. patent application Ser. No. 14/193,037),transposon-based, ZEN, meganuclease, or Mega-TAL, or Transposon-basedsystem. For example, PiggyBac (see Moriarty, B. S., et al., “Modularassembly of transposon integratable multigene vectors using RecWayassembly,” Nucleic Acids Research (8):e92 (2013) or sleeping beauty (seeAronovich, E. L, et al., “The Sleeping Beauty transposon system: anon-viral vector for gene therapy,” Hum. Mol. Genet., 20(R1): R14-R20.(2011) transposon systems can be used.

Site specific gene editing can also be achieved without homologousrecombination. An exogenous polynucleic acid can be introduced into acell genome without the use of homologous recombination. In some cases,a transgene can be flanked by engineered sites that are complementary toa targeted double strand break region in a genome. A transgene can beexcised from a polynucleic acid so it can be inserted at a double strandbreak region without homologous recombination.

Crispr System

Methods described herein can take advantage of a CRISPR system. Thereare at least five types of CRISPR systems which all incorporate RNAs andCas proteins. Types I, III, and IV assemble a multi-Cas protein complexthat is capable of cleaving nucleic acids that are complementary to theerRNA. Types I and III both require pre-erRNA processing prior toassembling the processed crRNA into the multi-Cas protein complex. TypesII and V CRISPR systems comprise a single Cas protein complexed with atleast one guiding RNA.

The general mechanism and recent advances of CRISPR system is discussedin Cong, L. et al., “Multiplex genome engineering using CRISPR systems,”Science, 339(6121): 819-823 (2013); Fu, et al., “High-frequencyoff-target mutagenesis induced by CRISPR-Cas nucleases in human cells,”Nature Biotechnology, 31, 822-826 (2013); Chu, V T et al. “Increasingthe efficiency of homology-directed repair for CRISPR-Cas9-inducedprecise gene editing in mammalian cells,” Nature Biotechnology 33,543-548 (2015); Shmakov, S. et al., “Discovery and functionalcharacterization of diverse Class 2 CRISPR-Cas systems,” Molecular Cell,60, 1-13 (2015); Makarova, K S et al., “An updated evolutionaryclassification of CRISPR-Cas systems,”, Nature Reviews Microbiology, 13,1-15 (2015). Site-specific cleavage of a target DNA occurs at locationsdetermined by both 1) base-pairing complementarity between the guide RNAand the target DNA (also called a protospacer) and 2) a short motif inthe target DNA referred to as the protospacer adjacent motif (PAM). Forexample, an engineered cell can be generated using a CRISPR system,e.g., a type II CRISPR system. A Cas enzyme used in the methodsdisclosed herein can be Cas9, which catalyzes DNA cleavage. Enzymaticaction by Cas9 derived from Streptococcus pyogenes or any closelyrelated Cas9 can generate double stranded breaks at target sitesequences which hybridize to 20 nucleotides of a guide sequence and thathave a protospacer-adjacent motif (PAM) following the 20 nucleotides ofthe target sequence.

Cas Protein

A vector can be operably linked to an enzyme-coding sequence encoding aCRISPR enzyme, such as a Cas protein (CRISPR-associated protein).Non-limiting examples of Cas proteins can include Cas 1, Cas 1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn 1 or Csx12),Cas 10, Csy1, Csy2, Csy3, Cse 1, Cse2, Csc 1, Csc2, Csa5, Csn2, Csm2,Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb 1, Csb2, Csb3,Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO,Csf4, Cpf1, c2c 1, c2c3, Cas9HiFi, homologues thereof, or modifiedversions thereof. An unmodified CRISPR enzyme can have DNA cleavageactivity, such as Cas9. A CRISPR enzyme can direct cleavage of one orboth strands at a target sequence, such as within a target sequenceand/or within a complement of a target sequence. For example, a CRISPRenzyme can direct cleavage of one or both strands within or within about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or morebase pairs from the first or last nucleotide of a target sequence. Avector that encodes a CRISPR enzyme that is mutated with respect to acorresponding wild-type enzyme such that the mutated CRISPR enzyme lacksthe ability to cleave one or both strands of a target polynucleotidecontaining a target sequence can be used. A Cas protein can be a highfidelity cas protein such as Cas9HiFi.

A vector that encodes a CRISPR enzyme comprising one or more nuclearlocalization sequences (NLSs), such as more than or more than about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, NLSs can be used. For example, a CRISPRenzyme can comprise more than or more than about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, NLSs at or near the ammo-terminus, more than or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, NLSs at or near the carboxyl-terminus, orany combination of these (e.g., one or more NLS at the ammo-terminus andone or more NLS at the carboxyl terminus). When more than one NLS ispresent, each can be selected independently of others, such that asingle NLS can be present in more than one copy and/or in combinationwith one or more other NLSs present in one or more copies.

Cas9 can refer to a polypeptide with at least or at least about 50%,60%, 70%, 80%, 90%, 100% sequence identity and/or sequence similarity toa wild type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes).Cas9 can refer to a polypeptide with at most or at most about 50%, 60%,70%, 80%, 90%, 100% sequence identity and/or sequence similarity to awild type exemplary Cas9 polypeptide (e.g., from S. pyogenes). Cas9 canrefer to the wild type or a modified form of the Cas9 protein that cancomprise an amino acid change such as a deletion, insertion,substitution, variant, mutation, fusion, chimera, or any combinationthereof.

A polynucleotide encoding an endonuclease (e.g., a Cas protein such asCas9) can be codon optimized for expression in particular cells, such aseukaryotic cells. This type of optimization can entail the mutation offoreign-derived (e.g., recombinant) DNA to mimic the codon preferencesof the intended host organism or cell while encoding the same protein.

CRISPR enzymes used in the methods can comprise NLSs. The NLS can belocated anywhere within the polypeptide chain, e.g., near the N- orC-terminus. For example, the NLS can be within or within about 1, 2, 3,4, 5, 10, 15, 20, 25, 30, 40, 50 amino acids along a polypeptide chainfrom the N- or C-terminus. Sometimes the NLS can be within or withinabout 50 amino acids or more, e.g., 100, 200, 300, 400, 500, 600, 700,800, 900, or 1000 amino acids from the N- or C-terminus.

An endonuclease can comprise an amino acid sequence having at least orat least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%,amino acid sequence identity to the nuclease domain of a wild typeexemplary site-directed polypeptide (e.g., Cas9 from S. pyogenes).

While S. pyogenes Cas9 (SpCas9) is commonly used as a CRISPRendonuclease for genome engineering, it may not be the best endonucleasefor every target excision site. For example, the PAM sequence for SpCas9(5′ NGG 3′) is abundant throughout the human genome, but a NGG sequencemay not be positioned correctly to target a desired gene formodification. In some cases, a different endonuclease may be used totarget certain genomic targets. In some cases, synthetic SpCas9-derivedvariants with non-NGG PAM sequences may be used. Additionally, otherCas9 orthologues from various species have been identified and these“non-SpCas9s” bind a variety of PAM sequences that could also be usefulfor the present invention. For example, the relatively large size ofSpCas9 (approximately 4 kb coding sequence) means that plasmids carryingthe SpCas9 cDNA may not be efficiently expressed in a cell. Conversely,the coding sequence for Staphylococcus aureus Cas9 (SaCas9) isapproximately 1 kilobase shorter than SpCas9, possibly allowing it to beefficiently expressed in a cell. Similar to SpCas9, the SaCas9endonuclease is capable of modifying target genes in mammalian cells invitro and in mice in vivo.

Alternatives to S. pyogenes Cas9 may include RNA-guided endonucleasesfrom the Cpf1 family that display cleavage activity in mammalian cells.Unlike Cas9 nucleases, the result of Cpf1-mediated DNA cleavage is adouble-strand break with a short 3′ overhang. Cpf1's staggered cleavagepattern may open up the possibility of directional gene transfer,analogous to traditional restriction enzyme cloning, which may increasethe efficiency of gene editing. Like the Cas9 variants and orthologuesdescribed above, Cpf1 may also expand the number of sites that can betargeted by CRISPR to AT-rich regions or AT-rich genomes that lack theNGG PAM sites favored by SpCas9.

Any functional concentration of Cas protein can be introduced to a cell.For example, 15 micrograms of Cas mRNA can be introduced to a cell. Inother cases, a Cas mRNA can be introduced from 0.5 micrograms to 100micrograms. A Cas mRNA can be introduced from 0.5, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100micrograms.

Guide RNA

As used herein, the term “guide RNA (gRNA)”, and its grammaticalequivalents can refer to an RNA which can be specific for a target DNAand can form a complex with a Cas protein. A guide RNA can comprise aguide sequence, or spacer sequence, that specifies a target site andguides an RNA/Cas complex to a specified target DNA for cleavage. Forexample, FIG. 15 , demonstrates that guide RNA can target a CRISPRcomplex to three genes and perform a targeted double strand break.Site-specific cleavage of a target DNA occurs at locations determined byboth 1) base-pairing complementarity between a guide RNA and a targetDNA (also called a protospacer) and 2) a short motif in a target DNAreferred to as a protospacer adjacent motif (PAM).

A method disclosed herein also can comprise introducing into a cell orembryo at least one guide RNA or nucleic acid, e.g., DNA encoding atleast one guide RNA. A guide RNA can interact with a RNA-guidedendonuclease to direct the endonuclease to a specific target site, atwhich site the 5′ end of the guide RNA base pairs with a specificprotospacer sequence in a chromosomal sequence.

A guide RNA can comprise two RNAs, e.g., CRISPR RNA (crRNA) andtransactivating crRNA (tracrRNA). A guide RNA can sometimes comprise asingle-guide RNA (sgRNA) formed by fusion of a portion (e.g., afunctional portion) of crRNA and tracrRNA. A guide RNA can also be adual RNA comprising a crRNA and a tracrRNA. A guide RNA can comprise acrRNA and lack a tracrRNA. Furthermore, a crRNA can hybridize with atarget DNA or protospacer sequence.

As discussed above, a guide RNA can be an expression product. Forexample, a DNA that encodes a guide RNA can be a vector comprising asequence coding for the guide RNA. A guide RNA can be transferred into acell or organism by transfecting the cell or organism with an isolatedguide RNA or plasmid DNA comprising a sequence coding for the guide RNAand a promoter. A guide RNA can also be transferred into a cell ororganism in other way, such as using virus-mediated gene delivery.

A guide RNA can be isolated. For example, a guide RNA can be transfectedin the form of an isolated RNA into a cell or organism. A guide RNA canbe prepared by in vitro transcription using any in vitro transcriptionsystem. A guide RNA can be transferred to a cell in the form of isolatedRNA rather than in the form of plasmid comprising encoding sequence fora guide RNA.

A guide RNA can comprise a DNA-targeting segment and a protein bindingsegment. A DNA-targeting segment (or DNA-targeting sequence, or spacersequence) comprises a nucleotide sequence that can be complementary to aspecific sequence within a target DNA (e.g., a protospacer). Aprotein-binding segment (or protein-binding sequence) can interact witha site-directed modifying polypeptide, e.g., an RNA-guided endonucleasesuch as a Cas protein. By “segment” it is meant a segment/section/regionof a molecule, e.g., a contiguous stretch of nucleotides in an RNA. Asegment can also mean a region/section of a complex such that a segmentmay comprise regions of more than one molecule. For example, in somecases a protein-binding segment of a DNA-targeting RNA is one RNAmolecule and the protein-binding segment therefore comprises a region ofthat RNA molecule. In other cases, the protein-binding segment of aDNA-targeting RNA comprises two separate molecules that are hybridizedalong a region of complementarity.

A guide RNA can comprise two separate RNA molecules or a single RNAmolecule. An exemplary single molecule guide RNA comprises both aDNA-targeting segment and a protein-binding segment.

An exemplary two-molecule DNA-targeting RNA can comprise a crRNA-like(“CRISPR RNA” or “targeter-RNA” or “crRNA” or “crRNA repeat”) moleculeand a corresponding tracrRNA-like (“trans-acting CRISPR RNA” or“activator-RNA” or “tracrRNA”) molecule. A first RNA molecule can be acrRNA-like molecule (targeter-RNA), that can comprise a DNA-targetingsegment (e.g., spacer) and a stretch of nucleotides that can form onehalf of a double-stranded RNA (dsRNA) duplex comprising theprotein-binding segment of a guide RNA. A second RNA molecule can be acorresponding tracrRNA-like molecule (activator-RNA) that can comprise astretch of nucleotides that can form the other half of a dsRNA duplex ofa protein-binding segment of a guide RNA. In other words, a stretch ofnucleotides of a crRNA-like molecule can be complementary to and canhybridize with a stretch of nucleotides of a tracrRNA-like molecule toform a dsRNA duplex of a protein-binding domain of a guide RNA. As such,each crRNA-like molecule can be said to have a correspondingtracrRNA-like molecule. A crRNA-like molecule additionally can provide asingle stranded DNA-targeting segment, or spacer sequence. Thus, acrRNA-like and a tracrRNA-like molecule (as a corresponding pair) canhybridize to form a guide RNA. A subject two-molecule guide RNA cancomprise any corresponding crRNA and tracrRNA pair.

A DNA-targeting segment or spacer sequence of a guide RNA can becomplementary to sequence at a target site in a chromosomal sequence,e.g., protospacer sequence) such that the DNA-targeting segment of theguide RNA can base pair with the target site or protospacer. In somecases, a DNA-targeting segment of a guide RNA can comprise from or fromabout 10 nucleotides to from or from about 25 nucleotides or more. Forexample, a region of base pairing between a first region of a guide RNAand a target site in a chromosomal sequence can be or can be about 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25nucleotides in length. Sometimes, a first region of a guide RNA can beor can be about 19, 20, or 21 nucleotides in length.

A guide RNA can target a nucleic acid sequence of or of about 20nucleotides. A target nucleic acid can be less than or less than about20 nucleotides. A target nucleic acid can be at least or at least about5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or morenucleotides. A target nucleic acid can be at most or at most about 5,10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.A target nucleic acid sequence can be or can be about 20 basesimmediately 5′ of the first nucleotide of the PAM. A guide RNA cantarget the nucleic acid sequence.

A guide nucleic acid, for example, a guide RNA, can refer to a nucleicacid that can hybridize to another nucleic acid, for example, the targetnucleic acid or protospacer in a genome of a cell. A guide nucleic acidcan be RNA. A guide nucleic acid can be DNA. The guide nucleic acid canbe programmed or designed to bind to a sequence of nucleic acidsite-specifically. A guide nucleic acid can comprise a polynucleotidechain and can be called a single guide nucleic acid. A guide nucleicacid can comprise two polynucleotide chains and can be called a doubleguide nucleic acid.

A guide nucleic acid can comprise one or more modifications to provide anucleic acid with a new or enhanced feature. A guide nucleic acid cancomprise a nucleic acid affinity tag. A guide nucleic acid can comprisesynthetic nucleotide, synthetic nucleotide analog, nucleotidederivatives, and/or modified nucleotides.

A guide nucleic acid can comprise a nucleotide sequence (e.g., aspacer), for example, at or near the 5′ end or 3′ end, that canhybridize to a sequence in a target nucleic acid (e.g., a protospacer).A spacer of a guide nucleic acid can interact with a target nucleic acidin a sequence-specific manner via hybridization (i.e., base pairing). Aspacer sequence can hybridize to a target nucleic acid that is located5′ or 3′ of a protospacer adjacent motif (PAM). The length of a spacersequence can be at least or at least about 5, 10, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 30 or more nucleotides. The length of a spacersequence can be at most or at most about 5, 10, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30 or more nucleotides.

A guide RNA can also comprises a dsRNA duplex region that forms asecondary structure. For example, a secondary structure formed by aguide RNA can comprise a stem (or hairpin) and a loop. A length of aloop and a stem can vary. For example, a loop can range from about 3 toabout 10 nucleotides in length, and a stem can range from about 6 toabout 20 base pairs in length. A stem can comprise one or more bulges of1 to about 10 nucleotides. The overall length of a second region canrange from about 16 to about 60 nucleotides in length. For example, aloop can be or can be about 4 nucleotides in length and a stem can be orcan be about 12 base pairs. A dsRNA duplex region can comprise aprotein-binding segment that can form a complex with an RNA-bindingprotein, such as a RNA-guided endonuclease, e.g., Cas protein.

A guide RNA can also comprise a tail region at the 5′ or 3′ end that canbe essentially single-stranded. For example, a tail region is sometimesnot complementarity to any chromosomal sequence in a cell of interestand is sometimes not complementarity to the rest of a guide RNA.Further, the length of a tail region can vary. A tail region can be morethan or more than about 4 nucleotides in length. For example, the lengthof a tail region can range from or from about 5 to from or from about 60nucleotides in length.

A guide RNA can be introduced into a cell or embryo as an RNA molecule.For example, a RNA molecule can be transcribed in vitro and/or can bechemically synthesized. A guide RNA can then be introduced into a cellor embryo as an RNA molecule. A guide RNA can also be introduced into acell or embryo in the form of a non-RNA nucleic acid molecule, e.g., DNAmolecule. For example, a DNA encoding a guide RNA can be operably linkedto promoter control sequence for expression of the guide RNA in a cellor embryo of interest. A RNA coding sequence can be operably linked to apromoter sequence that is recognized by RNA polymerase III (Pol III).

A DNA molecule encoding a guide RNA can also be linear. A DNA moleculeencoding a guide RNA can also be circular.

A DNA sequence encoding a guide RNA can also be part of a vector. Someexamples of vectors can include plasmid vectors, phagemids, cosmids,artificial/mini-chromosomes, transposons, and viral vectors. Forexample, a DNA encoding a RNA-guided endonuclease is present in aplasmid vector. Other non-limiting examples of suitable plasmid vectorsinclude pUC, pBR322, pET, pBluescript, and variants thereof. Further, avector can comprise additional expression control sequences (e.g.,enhancer sequences, Kozak sequences, polyadenylation sequences,transcriptional termination sequences, etc.), selectable markersequences (e.g., antibiotic resistance genes), origins of replication,and the like.

When both a RNA-guided endonuclease and a guide RNA are introduced intoa cell as DNA molecules, each can be part of a separate molecule (e.g.,one vector containing fusion protein coding sequence and a second vectorcontaining guide RNA coding sequence) or both can be part of a samemolecule (e.g., one vector containing coding (and regulatory) sequencefor both a fusion protein and a guide RNA).

A Cas protein, such as a Cas9 protein or any derivative thereof, can bepre-complexed with a guide RNA to form a ribonucleoprotein (RNP)complex. The RNP complex can be introduced into primary immune cells.Introduction of the RNP complex can be timed. The cell can besynchronized with other cells at G1, S, and/or M phases of the cellcycle. The RNP complex can be delivered at a cell phase such that HDR isenhanced. The RNP complex can facilitate homology directed repair.

A guide RNA can also be modified. The modifications can comprisechemical alterations, synthetic modifications, nucleotide additions,and/or nucleotide subtractions. The modifications can also enhanceCRISPR genome engineering. A modification can alter chirality of a gRNA.In some cases, chirality may be uniform or stereopure after amodification. A guide RNA can be synthesized. The synthesized guide RNAcan enhance CRISPR genome engineering. A guide RNA can also betruncated. Truncation can be used to reduce undesired off-targetmutagenesis. The truncation can comprise any number of nucleotidedeletions. For example, the truncation can comprise 1, 2, 3, 4, 5, 10,15, 20, 25, 30, 40, 50 or more nucleotides. A guide RNA can comprise aregion of target complementarity of any length. For example, a region oftarget complementarity can be less than 20 nucleotides in length. Aregion of target complementarity can be more than 20 nucleotides inlength.

In some cases, a dual nickase approach may be used to introduce a doublestranded break. Cas proteins can be mutated at known amino acids withineither nuclease domains, thereby deleting activity of one nucleasedomain and generating a nickase Cas protein capable of generating asingle strand break. A nickase along with two distinct guide RNAstargeting opposite strands may be utilized to generate a DSB within atarget site (often referred to as a “double nick” or “dual nickase”CRISPR system). This approach may dramatically increase targetspecificity, since it is unlikely that two off-target nicks will begenerated within close enough proximity to cause a DSB.

In some cases, a GUIDE-Seq analysis can be performed to determine thespecificity of engineered guide RNAs. The general mechanism and protocolof GUIDE-Seq profiling of off-target cleavage by CRISPR system nucleasesis discussed in Tsai, S. et al., “GUIDE-Seq enables genome-wideprofiling of off-target cleavage by CRISPR system nucleases,” Nature,33: 187-197 (2015).

A gRNA can be introduced at any functional concentration. For example, agRNA can be introduced to a cell at 10 micrograms. In other cases, agRNA can be introduced from 0.5 micrograms to 100 micrograms. A gRNA canbe introduced from 0.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, or 100 micrograms.

In some cases, a method can comprise an endonuclease selected from thegroup consisting of Cas 1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7,Cas8, Cas9, Cas 10, Csy 1, Csy2, Csy3, Cse 1, Cse2, Csc1, Csc2, Csa5,Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb 1,Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1,Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi, homologues thereof ormodified versions thereof. A Cas protein can be Cas9. In some cases, amethod can further comprise at least one guide RNA (gRNA). A gRNA cancomprise at least one modification. An exogenous TCR can bind a cancerneo-antigen.

Disclosed herein is a method of making an engineered cell comprising:introducing at least one polynucleic acid encoding at least oneexogenous LOV receptor sequence; introducing at least one guide RNA(gRNA) comprising at least one modification; and introducing at leastone endonuclease; wherein the gRNA comprises at least one sequencecomplementary to at least one endogenous genome. In some cases, amodification is on a 5′ end, a 3′ end, from a 5′ end to a 3′ end, asingle base modification, a 2′-ribose modification, or any combinationthereof. A modification can be selected from a group consisting of basesubstitutions, insertions, deletions, chemical modifications, physicalmodifications, stabilization, purification, and any combination thereof.

In accordance with an embodiment, the present invention provides atransformant transformed by the vectors described herein, including, forexample, mammalian cells, and specifically, pluripotent stem cells.

In some embodiments, the transformant is a stem cell. In otherembodiments, the transformant is a human derived stem cell.

In further embodiments, the transformant is a stem cell from a mammal,such as a pig, cow, sheep, goat, chicken or other livestock animal.

The invention further provides a host cell comprising any of therecombinant expression vectors described herein. As used herein, theterm “host cell” refers to any type of cell that can contain theinventive recombinant expression vector. The host cell can be aeukaryotic cell, e.g., plant, animal, fungi, or algae. The host cell canbe a cultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human or pig. The host cell can be an adherent cell ora suspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, stem cells. Insome preferred embodiments, the host cells are pluripotent stem cells ofhuman or porcine derivation.

Also provided by the invention is a population of cells comprising atleast one host cell described herein. The population of cells can be asubstantially homogeneous population, in which the population comprisesmainly of host cells (e.g., consisting essentially of) comprising therecombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein.

The host referred to in the inventive methods can be any host.Preferably, the host is a mammal. As used herein, the term “mammal”refers to any mammal, including, but not limited to, mammals of theorder Rodentia, such as mice and hamsters, and mammals of the orderLagomorpha, such as rabbits. It is preferred that the mammals are fromthe order Carnivora, including Felines (cats) and Canines (dogs). It ismore preferred that the mammals are from the order Artiodactyla,including Bovine (cows) and Swine (pigs) or of the order Perssodactyla,including Equine (horses). It is most preferred that the mammals are ofthe order Primates, Ceboids, or Simoids (monkeys) or of the orderAnthropoids (humans and apes). An especially preferred mammal is thepig.

Stem cells for use in the methods disclosed herein are not limited inany way and may be obtained from any source. Preferably the stem cellsare pluripotent stem cells that are obtained from a mammalian source.

The term “nucleic acid” as used herein, includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means anisolated or purified polymer of DNA or RNA, which can be single-strandedor double-stranded, synthesized or obtained (e.g., isolated and/orpurified) from natural sources, which can contain natural, non-naturalor altered nucleotides, and which can contain a natural, non-natural oraltered internucleotide linkage, such as a phosphoroamidate linkage or aphosphorothioate linkage, instead of the phosphodiester found betweenthe nucleotides of an unmodified oligonucleotide. In some embodiments,the nucleic acid does not comprise any insertions, deletions,inversions, and/or substitutions. However, it may be suitable in someinstances, as discussed herein, for the nucleic acid to comprise one ormore insertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3^(rd)ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, NY, 1994. For example, anucleic acid can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed upon hybridization (e.g.,phosphorothioate derivatives and acridine substituted nucleotides).Examples of modified nucleotides that can be used to generate thenucleic acids include, but are not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The invention also provides an isolated or purified nucleic acidcomprising a nucleotide sequence which is complementary to thenucleotide sequence of any of the nucleic acids described herein or anucleotide sequence which hybridizes under stringent conditions to thenucleotide sequence of any of the nucleic acids described herein.

The term “isolated” as used herein means having been removed from itsnatural environment. The term “purified” as used herein means havingbeen increased in purity, wherein “purity” is a relative term, and notto be necessarily construed as absolute purity. For example, the puritycan be at least about 50%, can be greater than 60%, 70% or 80%, or canbe 100%.

The term “primer” refers to an oligonucleotide, whether natural orsynthetic, capable of acting as a point of initiation of DNA synthesisunder conditions in which synthesis of a primer extension productcomplementary to a nucleic acid strand is induced, i.e., in the presenceof four different nucleoside triphosphates and an agent forpolymerization (i.e., DNA polymerase or reverse transcriptase) in anappropriate buffer and at a suitable temperature. A primer is preferablya single-stranded oligodeoxyribonucleotide. The appropriate length of aprimer depends on the intended use of the primer but typically rangesfrom about 10 to about 30 nucleotides. Short primer molecules generallyrequire cooler temperatures to form sufficiently stable hybrid complexeswith the template. A primer need not reflect the exact sequence of thetemplate but must be sufficiently complementary to specificallyhybridize with a template. When primer pairs are referred to herein, thepair is meant to include one forward primer which is capable ofhybridizing to the sense strand of a double-stranded target nucleic acid(the “sense primer”) and one reverse primer which is capable ofhybridizing to the antisense strand of a double-stranded target nucleicacid (the “antisense primer”).

“Probe” refers to an oligonucleotide which binds through complementarybase pairing to a sub-sequence of a target nucleic acid. A primer may bea probe. It will be understood by one of skill in the art that probeswill typically substantially bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are typically directly labeled(e.g., with isotopes or fluorescent moieties) or indirectly labeled suchas with biotin to which a streptavidin complex may later bind. Byassaying for the presence or absence of the probe, one can detect thepresence or absence of the target, by Southern blot for example.

The recombinant expression vectors of the invention can be preparedusing standard recombinant DNA techniques described in, for example,Sambrook et al., supra, and Ausubel et al., supra. Constructs ofexpression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColEl, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

In some exemplary embodiments, the CRISPR system has been used in thissystem to permanently incorporate the opto-FGFR system into the humanstem cells. We specifically target the AAVS1 locus (a safe-harbor regionwith constitutive expression).

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA or RNA based.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected hosts. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like. Suitable marker genes for the inventiveexpression vectors include, for instance, neomycin/G418 resistancegenes, hygromycin resistance genes, histidinol resistance genes,tetracycline resistance genes, puromycin resistance genes and ampicillinresistance genes.

The selection of promoters, e.g., strong, weak, inducible,tissue-specific and developmental-specific, is within the ordinary skillof the artisan. Similarly, the combining of a nucleotide sequence with apromoter is also within the skill of the artisan. The promoter can be anon-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV)promoter, the tet-on promoter, or the ubiquitin C promoter, for example.

Fibroblast growth factor (FGF) free pluripotent stem cell culturesystems.

Currently, there are a few prior art culture optogenetics systems on themarket, but these systems are all very expensive. High cost commercialproducts prevent large volume optogenetics studies and thus, limit therate of research in this field. There is also a lack of instrumentationthat allows for precise control of illumination and remote monitoring ofmultiple culture plates.

In accordance with some embodiments, the fibroblast growth factor freepluripotent stem cell culture system of the present invention is ahardware, software, and mobile application system capable of precisecontrol and real-time monitoring of cell culture illumination that canserve as an inexpensive alternative to high cost commercial products.The inventive illumination system is designed to be easy-to-assemble,easy-to-use, and low-cost, utilizing 3D printing of thermoplasticmaterials, programmable open-source electronics, and otherreadily-available electronic parts. Additionally, the inventiveillumination system was designed to be adjustable and be customized tothe individual researcher. The 3D models and source code are publiclyavailable to be edited for any specific needs. Additionally, the LEDilluminating device of the inventive illumination system can becustomized with any color LED to be used with any photosynthetic proteinof interest. The inventive illumination system provides the opportunityto apply a flexible optical culture system to cell cultures in anylaboratory.

As such, in accordance with an embodiment, the present inventionprovides a fibroblast growth factor free pluripotent stem cell culturesystem comprising one or more transformed cells as described herein andan illumination source.

In accordance with an embodiment, the present invention provides a cellculture illumination system comprising a cell culture substrate, anupper and lower enclosure, one or more illumination sources, one or morecircuit boards, a power source, and a microcontroller.

Referring now to FIG. 16 , in general, the cell culture illuminationsystem 100 is designed in such a way that it is easily implemented, forexample for use with standard size cell culture plates. The illuminationsystem comprises one or more illumination sources 101 of any type whichare capable of irradiating light of a tunable frequency into each well,and to the cells contained within each well of a cell culture plate 106.In some embodiments the illumination source is one or more tunable LEDs.The illumination sources are connected electronically to at least onecircuit board 102 and are arranged inside an upper enclosure 104 whichis designed to accept the cell culture plate. The circuit board 102 isconnected electronically to a microcontroller 103 which controls thelight frequency and intensity. The microcontroller 103 and upperenclosure 104 are designed to fit into a lower enclosure 105 and can beheld together with fasteners such as screws. The microcontroller 103 iscoupled to a power source 107 (see FIG. 17 ) such as a USB port from acomputer. Other power sources could also be used, including for example,wired to a common power outlet or that can be re-chargeable such as abattery.

As shown in FIGS. 16-18 , the microcontroller 103 is connected either bywire or wirelessly to a main computer server 200. The main computerserver 200 can be programmed with information related to theillumination frequency, intensity and duration.

It should be noted that the microcontroller 103 and the main computerserver 200 can all include a computing device such as a microprocessor,hard drive, solid state drive, smartphone or any other suitablecomputing device known to or conceivable by one of skill in the art. Asshown in FIG. 18 , the main computer server 200 can be controlled by acomputing client device 203, or a smartphone or mobile computing device202 which is programmed with a non-transitory computer readable mediumthat is programmed with steps to execute the different illuminationfrequencies, intensities, and durations of irradiation.

Any such computer application will be fixed on a non-transitory computerreadable medium. It should be noted that the computer application isprogrammed onto a non-transitory computer readable medium that can beread and executed by any of the computing devices mentioned in thisapplication. The non-transitory computer readable medium can take anysuitable form known to one of skill in the art. The non-transitorycomputer readable medium is understood to be any article of manufacturereadable by a computer or smartphone. Such non-transitory computerreadable media includes, but is not limited to, magnetic media, such asfloppy disk, flexible disk, hard, disk, reel-to-reel tape, cartridgetape, cassette tapes or cards, optical media such as CD-ROM, DVD,blu-ray, writable compact discs, magneto-optical media in disc, tape, orcard form, and paper media such as punch cards or paper tape.Alternately, the program for executing the method and algorithms of thepresent invention can reside on a remote server or other networkeddevice. Any databases associated with the present invention can behoused on a central computing device, server(s), in cloud storage, orany other suitable means known to or conceivable by one of skill in theart. All of the information associated with the application istransmitted either wired or wirelessly over a network, via the internet,cellular telephone network, or any other suitable data transmissionmeans known to or conceivable by one of skill in the art.

In accordance with an embodiment, the present invention provides amethod for maintaining a pluripotent stem cell line in culturecomprising: a) a plurality of pluripotent stem cells in a fibroblastgrowth factor free culture medium, wherein said are transformed with theexpression vectors described herein; b) said transformed cells aremaintained in the cell culture system as described herein; and c)periodically irradiating the cells of b) at a wavelength of about 470 nmfor a sufficient time and intensity such that the cells maintain theirpluripotent capability.

In some exemplary embodiments the intensity at the maximal output ineach well of a 6-well plate was between 1 and 23 μW/mm², with theability to vary the intensity from 2, 3, 4, 5, 10, 15, 20 or greaterμW/mm². The LEDs can be illuminated for different lengths of time (forexample, but without limitation, 0 to 10 min at 1 μW/mm²) and intensity(for example, but without limitation, 0 to 1 μW/mm² for 5 min). For thelong term maintenance of Opto-FGFR hPSCs, the blue light illuminationcan be repeated 1 min, 5 min, or 10 min or longer in every 1-10 hours,preferably about 2 hours at 1 μW/mm² during cell culture.

The present inventive approaches defined herein for the opticalmaintenance of human and porcine PSCs provides new insights for analternative synthetic approach for the next generation in stem cellresearch. Furthermore, considering the number of signaling pathways thatare crucial for controlling cell specification processes, the opto-FGFRPSCs of the present invention are a proof-of-concept for the opticalcontrol over a plethora of mammalian stem cell fates.

The following examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Without further elaboration, it is believed that one skilled in the art,using the preceding description, can utilize the present invention tothe fullest extent. The following examples are illustrative only, andnot limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices, and/or methods described andclaimed herein are made and evaluated, and are intended to be purelyillustrative and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.) butsome errors and deviations should be accounted for herein. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees Celsius or is at ambient temperature, and pressure is at or nearatmospheric. There are numerous variations and combinations of reactionconditions, e.g., component concentrations, desired solvents, solventmixtures, temperatures, pressures and other reaction ranges andconditions that can be used to optimize the product purity and yieldobtained from the described process. Only reasonable and routineexperimentation will be required to optimize such process conditions.

Materials and Methods

Pluripotent cell line culture. The human embryonic stem cells (H9,WiCell) and human induced pluripotent stem cells (induced from GM00024C,fibroblasts from Coriell) were cultured using standard protocols [26].The hESCs and hiPSCs were cultured on mouse embryonic fibroblasts (MEFs,Applied StemCell) and pre-plated at 12,000 to 15,000 cells/cm2. Themedium contained DMEM/F12, 20% knockout serum replacement, 1 mML-glutamine, 100 μM MEM non-essential amino acids, and 0.1 mMβ-mercaptoethanol (Life Technologies). Ten ng/mL of FGF2 (R&D Systems)was added after sterile filtration, and the cells were fed daily andpassaged weekly using either 6 U/mL Dispase (Life Technologies) ormechanical means. For feeder-free culture conditions, hPSCs werecultured onto recombinant human Laminin 521 (Life Technologies)-coatedplates with StemFit® Basic02 medium (Ajinomoto). The porcine inducedpluripotent stem cells (induced from P350-05; PCASMC, porcine coronaryartery smooth muscle cells from Sigma-Aldrich) were cultured withmitotically inactivated MEFs in the PSC medium, and the cells were feddaily and passaged weekly using either 6 U/mL Dispase or mechanicalmeans. For routine cell culture, all experiments including passagingwere performed in a dark culture room with safelight that does notaffect LOV photodimerization.

Plasmid construct. To establish the Opto-FGFR hESC and hiPSC lines, theEGFP region of the AAV-CAGGS-EGFP vector (Addgene plasmid #22212) wasreplaced by a region of the opto-FGFR1 domain. The PHR domain from thecytoplasmic FGFR1-Cry2 PHR-mCitrine vector and the LOV domain from themFGFR1-LOV vector (Addgene plasmid #58745) were used for theconstruction of AAV-opto-FGFR. AAV-opto-FGFRs were then used toestablish the opto-FGFR targeted to the AAVS1 region. Two guide RNAs(gRNAs) sequences for opto-FGFR knock-in at the pRosa26 region werechosen to target the first intron of pRosa26 and subcloned into gRNACloning Vector (Addgene plasmid #41824) as described previously [27].The sequences for pRosa26 gRNA constructs were as follows: pRosa26-gRNA#1,5′-CCTGGCTTAACCTGATTCTT-3′ (SEQ ID NO:10); pRosa26-gRNA #91,50-GTGAGAGTTATCTGACCGTA-3′ (SEQ ID NO:11). All insert sequences wereverified by Sanger DNA sequencing (JHU Synthesis & Sequencing Facility).

TABLE 2 Sequences of Primer Used in This Study Genes Forward ReverseDPPA3 5′-TTAATCCAACCTA 5′-AGGGGAAACAGAT CATCCCAGGG-3′ TCGCTACTA-3′(SEQ ID NO: 12) (SEQ ID NO: 13) ESG1 5′-ATATCCCGCCGTG 5′-ACTCAGCCATGGAGGTGAAAGTTC-3′ CTGGAGCATCC-3′ (SEQ ID NO: 14) (SEQ ID NO: 15) FOXA25′-CGACTGGAGCAGC 5′-TACGTGTTCATGC TACTATGC-3′ CGTTCAT-3′ (SEQ ID NO: 16)(SEQ ID NO: 17) GAPDH 5′-CGAGATCCCTCCA 5′-TTCTAGACGGCAG AAATCAA-3′GTCAGGT-3′ (SEQ ID NO: 18) (SEQ ID NO: 19) KLF4 5′-TCCCATCTTTCTC5′-GGATCGGATAGGT CACGTTC-3′ GAAGCTG-3′ (SEQ ID NO: 20) (SEQ ID NO: 21)MSGN1 5′-AGCGAAGGCTGCA 5′-TGGCCTCTCTGGC GTGTC-3′ TGTAGAC-3′(SEQ ID NO: 22) (SEQ ID NO: 23) NANOG 5′-CATGAGTGTGGAT 5′-CCTGAATAAGCAGCCAGCTTG-3′ ATCCATGG-3′ (SEQ ID NO: 24) (SEQ ID NO: 25) OCT45′-AGTGAGAGGCAAC 5′-ACACTCGGACCAC CTGGAGA-3′ ATCCTTC-3′ (SEQ ID NO: 26)(SEQ ID NO: 27) REX1 5′-CAGATCCTAAACA 5′-GCGTACGCAAATT GCTCGCAGAAT-3′AAAGTCCAGA-3′ (SEQ ID NO: 28) (SEQ ID NO: 29) PAX6 5′-CTTTGCTTGGGAA5′-AGCCAGGTTGCGA ATCCGAG-3′ AGAACTC-3′ (SEQ ID NO: 30) (SEQ ID NO: 31)SOX1 5′-CCTCGGATCTCTG 5′-GCAGGTACATGCT GTCAAGT-3′ GATCATCTC-3′(SEQ ID NO: 32) (SEQ ID NO: 33) SOX2 5′-GGGAAATGGGAGG 5′-TTGCGTGAGTGTGGGTGCAAAAGAGG-3′ GATGGGATTGGTG-3′ (SEQ ID NO: 34) (SEQ ID NO: 35) SOX175′-CGCACGGAATTTG 5′-GGATCAGGGACCT AACAGTA-3′ GTCACAC-3′ (SEQ ID NO: 36)(SEQ ID NO: 37) TBX6 5′-AAGTACCAACCCC 5′-TAGGCTGTCACGG GCATACA-3′AGATGAA-3′ (SEQ ID NO: 38) (SEQ ID NO: 39) pGapdh 5′-CTCAACGGGAAGC5′-CCCTGTTGCTGTA TCACTG-3′ GCCAAAT-3′ (SEQ ID NO: 40) (SEQ ID NO: 41)pGata4 5′-GCTTCGCGGGCTC 5′-CCGGTTGATGCCA CTACT-3′ TTCAT-3′(SEQ ID NO: 42) (SEQ ID NO: 43) pLin28a 5′-TGCCGGCATCTGT5′-GCAGTTTGCATTC AAATGGT-3′ CTTGGCA-3′ (SEQ ID NO: 44) (SEQ ID NO: 45)pMsgn1 5′-CGCTGGAGTCCTA 5′-GTCTGTGAGTTCC TTCTTCG-3′ CCGATGT-3′(SEQ ID NO: 46) (SEQ ID NO: 47) pNanog 5′-TTGCCCCGAAGCA 5′-CCAGCTCTGATTATCCATT-3′ CCCCACA-3′ (SEQ ID NO: 48) (SEQ ID NO: 49) pPax65′-AGTTCTTCGCAAC 5′-CATTTGGCCCTTC CTGGCTA-3′ GATTAGA-3′ (SEQ ID NO: 50)(SEQ ID NO: 51) pOct4 5′-TGAGGCTTTGCAG 5′-ACTGCTTGATCGT CTCAGTT-3′TTGCCCT-3′ (SEQ ID NO: 52) (SEQ ID NO: 53) pSox1 5′-CACCCGGATTACA5′-GAGTTGGAGATGG AGTACCG-3′ GGCTGTA-3′ (SEQ ID NO: 54) (SEQ ID NO: 55pSox2 5′-TAAGTACACACTG 5′-CATGGAACCGAG CCCGGAG-3′ CGTCATGC-3′(SEQ ID NO: 56) (SEQ ID NO: 57) pSox17 5′-TCGGGGACATGAA 5′-GCGGCCGGTACTTGATGAAG-3′ GTAGTT-3′ (SEQ ID NO: 58) (SEQ ID NO: 59) pTbx65′-AGTATCAGCCCCG 5′-CTGCTCGGGATCT CATACAC-3′ GACTCTC-3′ (SEQ ID NO: 60)(SEQ ID NO: 61)

Gene targeting. The hESC, hiPSC, and piPSC lines were cultured in Rhokinase (ROCK) inhibitor (Calbiochem) for 24 h before nucleofection [28].The cells were harvested using Accutase (Innovative Cell Technologies),and 2×10⁶ cells were resuspended in the appropriate reagent (Lonza) with1 μg Cas9 plasmid (Addgene plasmid #41815), 1 μg guide RNA (Addgeneplasmid #41817, for opto-FGFR knock-in at AAVS1 locus; pRosa26-gRNA #1or #91, for opto-FGFR knock-in at pRosa26 locus), and 1 μg donorplasmid. Nucleofection was performed according to the manufacturer'sinstructions (Lonza). The cells were subsequently plated onpuro-resistant MEFs (Applied StemCell), and puromycin was used forcolony selection. The puromycin-resistant colonies were manually pickedand expanded.

Blue light illumination. A customized blue light illumination plate(TouchBright W-Series) was designed and manufactured by Live CellInstrument (Seoul, Korea). This plate contained 42 individual LEDs (70mW per LED) per well on a 6-well plate. The light intensity wascontrolled by software (Live Cell Instrument), and the actual lightintensity at 470 nm to the cell plate was measured by LaserCheck(Coherent). The intensity at the maximal output in each well of a 6-wellplate was 23 μW/mm². The LEDs were illuminated for different lengths oftime (0-10 min. at 1 μW/mm²) and intensity (0-1 μW/mm² for 5 min). Forthe long-term maintenance of Opto-FGFR hPSCs with blue light during thepassaging, the blue light illumination was repeated at 1, 5, or 10 min.every 2 h at 1 μW/mm² during cell culture. The DIY LED illuminatingdevice contained 1 individual LED (60 mW per LED) per well on a 6- or12-well plate. The maximal output in each well of a 6-well plate was 7ρW/mm².

Western blot analysis. The phosphorylation status of MEK and ERK1/2 weredetermined by Western blot analysis. The cells were starved from FGF2protein for 24 h, and then exposed to either FGF2 protein or blue lightillumination. One μg aliquots of whole cell lysates were loaded onto4-20% gradient SDS-PAGE gels and then transferred to nitrocellulosemembranes (Bio-Rad) using the Trans-Blot® Turbo™ Transfer System(Bio-Rad). The detection of phosphorylated ERK1/2 proteins (42 and 44kDa) was performed using a phospho-ERK1/2 antibody (Cell Signaling),total ERK1/2 expression level was determined using a totalphospho-ERK1/2 antibody (Cell Signaling), detection of pMEK protein (45kDa) was performed using a pMEK1/2 antibody (Cell Signaling), and totalMEK expression level was determined using a total MEK1/2 antibody (CellSignaling). The expression levels are quantified by normalizing to GAPDHexpression (Cell Signaling). Following the primary antibody incubation,the membranes were incubated with the appropriate secondary antibodiesand subsequently visualized with an ECL kit (Pierce).

Transcriptome analysis. Briefly, total RNA was extracted from cellpellets using TRIzol Reagent (Life Technologies), and 1 μg of RNA wasreverse transcribed using the High Capacity cDNA Reverse TranscriptionKit (Applied Biosystems). The qRT-PCR mixtures were prepared with SYBRGreen PCR Master Mix (Kapa Biosystem), and the reactions were performedusing the Mastercycler ep Realplex2 (Eppendorf). The transcriptionlevels were assessed by normalizing to GAPDH expression. The RNA-seqlibraries were constructed using TruSeq Stranded Total RNA RiboZero Goldsample Prep Kit (Illumina) following the manufacturer's protocol. Afterthe sequencing run, the Illumina Real Time Analysis (RTA) module wasused to perform image analysis and base calling, and the BCL ConverterSoftware (bcl2fastq v2.17.1.14) was used to generate FASTQ files thatcontained the sequence reads. The pair-end reads of cDNA sequences werealigned back to the human genome (UCSC hg19 from Illumina iGenome) bythe spliced read mapper TopHat (v2.0.9). The sequencing depth is over 90million (45 million paired-ends) mappable sequencing reads. Thealignment statistics and Q/C was achieved by SAMtools (v0.1.18) andRSeQC (v2.3.5) to calculate quality control metrics on the resultingaligned reads; this provides information on the mappability, uniformityof gene body coverage, insert length distributions, and junctionannotation. The heatmap from the RNA-seq data was generated followed themanufacturer's instructions (Partek® Genomics Suite®, version 6.6). Tocreate the heatmap, the undifferentiated-experimental anddifferentiated-control groups were compared to generate the 8132differentially expressed gene set, which was corrected to a fold changeof greater or less than two and a false discovery rate (FDR) with apvalue <0.05. Next, a clustering analysis of this heatmap was performedusing the hierarchical clustering method with standardized expressionnormalization (a default process in the Partek® Genomics Suite®, version6.6). The single-cell qRT-PCR was performed as previously described[29]. Briefly, OCT4::EGFP+single-cells were isolated byfluorescence-activated cell sorting (FACS) into a 96-well platecontaining 10 μg of lysis buffer. These plates were then sealed forsingle-cell reverse transcription and cDNA amplification using auniversal sequence from the adaptor. The final amplified cDNA was usedfor qRT-PCR.

FACS analysis. The cells were dissociated with Accutase for flowcytometry and analyzed using BD FACSCalibur (Becton Dickinson), and thedata was visualized using FlowJo software (Tree Star Inc.).

In vitro differentiation into three germ layers. Opto-FGFR hPSCscultured with blue light for more than 6 weeks were differentiated intothree germ layer lineages following the manufacturer's protocol(STEMdiff™ Trilineage Differentiation Kit, Catalog #05230), and theimmunofluorescent staining and RT-PCR for the representative markers ofeach lineage were performed at the recommended time point.

Directed differentiation of hPSCs. Undifferentiated hPSCs weredissociated into single cells using Accutase to differentiate intodesired cell types. For directed differentiation of hPSC-derivedmyotubes and hPSC-derived dopaminergic neurons, we followed previouslydescribed protocols [26,30].

Teratoma formation assay. The teratomas were generated by theintramuscular injection of Opto-FGFR hESCs and Opto-FGFR hiPSCs culturedwith blue light for more than 8 weeks into NOD SCID mice. In brief,5×10⁶ cells were harvested and injected into the hindlimb of 6-week-oldmale NOD SCID mice. Approximately 7 weeks after injection, the tumorswere dissected and fixed in 10% formalin (Sigma-Aldrich); the paraffinsections were prepared and stained with hematoxylin/eosin. Thedifferentiated tissues that represent the three embryonic germ layerswere identified.

LED illuminating device and software. The LED illuminating deviceconsists of 6 or 12 LED diodes (EDGELEC), a printed circuit board(Elegoo), a microcontroller (Elegoo), and a 3D printed plastic casing.The LEDs were soldered onto the circuit board, which was wired to themicrocontroller. The plastic casing was designed using computer-aideddesign software (AutoCAD) and printed with a 3D plastic printer(Monoprice 3D Select) using polylactic acid plastic (HATCHBOX). Thecasing was secured together using metal screws (Snug Fastener). Thecomponents for the LED illuminating device should be accurately printedand assembled to fix the distance between the cells and the light, amongthe devices. The software to control the LED illuminating device and themobile application were developed in Processing 3.5.3.

Statistical analysis. All statistical analyses were performed usingPrism 6 (GraphPad). The values were the results of at least threeindependent experiments, with multiple replicates of each, and reportedas the mean±SEM. Differences between two samples were analyzed forsignificance using the unpaired two-tailed Student's t-test in Prism(GraphPad).

Results Generation of a Novel FGF2-Free Human PSC Culture System

Generation of a light-inducible FGF signaling system. To create alight-inducible FGF signaling system for maintaining the pluripotency ofPSCs, the intracellular domain of FGFR1 was fused with aphotoactivatable LOV domain (discovered in Vaucheria fridida) and amyristoylation signal peptide (Myr) [31-35] (FIGS. 1A and B). Thisopto-FGFR cassette was inserted into the AAVS1 safe-harbor locus inOCT4::EGFP human embryonic stem cells (hESCs) [36] and human iPSCs(hiPSCs) using the CRISPR/Cas9 system [27,37] (FIG. 1C and FIG. 6A;Opto-FGFR hESC/hiPSC lines). The isolated Opto-FGFR hESC/hiPSC clonesexhibited a typical colony morphology and expressed the pluripotencymarker NANOG (FIGS. 6B and 6C). To test the effects of blue lightillumination on the established Opto-FGFR hPSCs (FIG. 6D), we comparedthe phosphorylation levels of ERK1/2 and MEK after treatment with FGF2protein or blue light (470 nm) illumination because phosphorylation ofMEK and ERK1/2 are markers for activation of the FGF signaling pathwayin hESCs [12,15]. After 24 h of FGF2 protein starvation, the cells wereharvested at 15 min. intervals up to 120 min. following 10 ng/mL of FGF2supplementation. For the blue light illumination, cells were collectedat the same time points following 1 μW/mm² of blue light illuminationfor 5 min. Western blot analyses demonstrated activated FGF signaling inboth conditions (FIGS. 6E and 6F).

Optimization of the optical induction of FGF signaling. Next, weoptimized the light illumination conditions and found that 5 min. ofillumination was sufficient to induce FGF signaling, and the blue lightexposure resulted in faster activation of FGF signaling than FGF2supplementation (FIGS. 6E and 6F). The effects of the different exposuretimes (0-10 min. at 1 ρW/mm²) and intensities of blue light (0-1 μW/mm²for 5 min) on ERK1/2 phosphorylation were measured, and the resultsshowed the saturation and tunability of blue light in opto-FGFR (FIG. 7). In addition, we found that the blue light illumination-basedactivation of FGF signaling in Opto-FGFR hESCs showed similar levels ofprotein phosphorylation in multiple downstream targets as those treatedwith recombinant FGF2 protein (FIG. 8 ). These data suggest that theoptical induction of FGF signaling via the LOV module has effectscomparable to the activation via FGF2 protein supplementation in hPSCculture.

Long-term validation of the light-inducible FGF signaling system. TheFGF2 protein is a critical medium component for maintaining theundifferentiated state in hPSCs; however, in the daily feedingcondition, its concentration decreased by approximately 40% in 4 h andapproximately 10% in 24 h after the initial feeding [38] (FIG. 1D, +F,positive control; FGF2 protein treatment). To repeatedly stimulate theFGF signaling at the appropriate level, we maintained Opto-FGFR hPSCswith a pulsed (not continued) blue light (FIG. 1D, +L, blue lightillumination; 1 ρW/mm², illumination of blue light for 1 min. or 5 min.in every 2 h). While the Opto-FGFR hESCs cultured in the dark were fullydifferentiated after 2 weeks, the cells cultured with pulsed blue lightmaintained hESC-colony morphologies for more than 1 year without FGF2supplementation (FIG. 1D). The optically maintained hESCs from multiplepassages showed a normal karyotype and comparable expression levels ofpluripotency markers, including OCT4 and NANOG mRNAs and OCT4, NANOG,and SSEA4 proteins, to that of the cells maintained with FGF2supplementation (FIGS. 1F and 1G and FIG. 9 ). Notably, the Opto-FGFRhiPSCs optically maintained for 12 weeks still expressed multiplepluripotency markers, OCT4 and NANOG mRNAs, and TRA-1-81 and NANOGproteins (FIGS. 1G and 1H). Moreover, the Opto-FGFR hESCs maintainedwith blue light illumination were successfully recovered after afreeze/thaw cycle (FIG. 10 ). To compare the transcriptionaldistribution of key pluripotency markers in hPSCs maintained with eitherpulsed blue light or FGF2 protein in more detail, we further profiledthe gene expression of pluripotency markers at a single-cell level. Thecells maintained with either condition for over 45 weeks were purifiedto OCT4::EGFP+single-cells by cell sorting (FIG. 11A). The expressionlevels of key pluripotency marker genes, such as OCT4, NANOG, SOX2,REX1, ESG1, and KLF4, in the optically maintained single-PSCs weresimilar to the levels in the single-PSCs maintained with FGF2supplementation (FIG. 11B). These data demonstrate that our opticalinduction system for FGF signaling is a suitable long-term culturesystem for maintaining the pluripotency of hPSCs.

Conservation of pluripotency in optically maintained hPSC. Toinvestigate the similarities at the molecular level between the lightillumination and FGF2 treatment conditions that could conserve thepluripotency of hPSCs, we employed global transcriptome analyses(RNA-seq) (FIG. 12 ). The hierarchical clustering analysis demonstratedthat the optically maintained hESCs (1, 5, or 10 min. of blue lightillumination every 2 h for 3 weeks) had comparable transcriptionalcharacteristics to that of the cells maintained with FGF2supplementation, but not to that of the differentiated cells (FIGS. 2Aand 2B). Moreover, the expression levels of pluripotency marker genes inthe optically maintained hPSCs were similar to the levels in the hPSCsmaintained with FGF2 supplementation (FIG. 2C). The majority ofsignificantly differentially expressed genes either in the opticallymaintained hPSCs or in the hPSCs maintained with FGF2 protein overlappedwith those of the differentiated cells (FIG. 2D). In a gene ontology(GO) analysis, the number of stem cell maintenance-related GO terms wassignificantly over-represented in the upregulated genes of the opticallymaintained hESCs over the differentiated cells (FIG. 2E), whichemphasizes that illumination is sufficient to maintain stem cellpluripotency. In contrast, various cell differentiation-related GO termswere significantly enriched in the downregulated genes of the opticallymaintained hESCs over the differentiated cells (FIG. 2F), which confirmsthat the light illumination condition can maintain the transcriptionalprofiles of human pluripotency similar to that of the FGF2 treatmentgroup and also be sufficient to prevent any spontaneous differentiationof PSCs.

Preserved pluripotency in optically maintained hPSC. To find the directdifferentiation potentials of hPSCs optically maintained, wedifferentiated these cells into three germ layers (FIG. 3A).Quantitative real-time PCR (qRT-PCR) results showed that the expressionlevels of PAX6 and SOX/(ectoderm), TBX6 and MSGN1 (mesoderm), and FOXA2and SOX/7 (endoderm) were significantly increased in the differentiatedOpto-FGFR hPSCs compared to the undifferentiated cells (FIG. 3B, 3F).The differentiation abilities were also confirmed by immunostaining withNESTIN (ectoderm), TBX6 (mesoderm), and FOXA2 (endoderm) antibodies(FIG. 3C). We further verified the directed differentiation abilities ofoptically maintained hPSCs into TH+/TJU1+dopaminergic neuron (ectoderm)or MF20+skeletal muscle (mesoderm) (FIG. 3D). Additionally, the in vivodifferentiation capacity of Opto-FGFR hPSCs was confirmed by a teratomaformation assay. The three germ layers were observed in teratoma tissuevia hematoxylin/eosin staining (FIG. 3E, 3G). These results showed thatlong-term culture conditions along with the optical induction of the FGFsignaling pathway maintained the pluripotency of PSCs without losing thecell specification capability toward the three germ layers.

Generation of a novel FGF2-free porcine PSC culture system. Porcine PSCsalso show an FGF2-dependency when maintaining their pluripotencies[17,18]. To expand our opto-FGFR system to piPSCs, the opto-FGFRcassette was inserted into the porcine Rosa26 (pRosa26) safe-harborlocus in piPSCs using the CRISPR/Cas9 system (FIG. 4A). For theopto-FGFR knock-in at pRosa26 locus, two gRNAs were designed and appliedto target the first intron of pRosa26 [39], and the opto-FGFR knock-inefficiencies were comparable with each other (FIG. 13A). The selectedOpto-FGFR piPSC line showed a typical colony morphology and enabled theoptical activation of the FGF signaling pathway (FIGS. 13B and 13C).While the Opto-FGFR piPSCs cultured with pulsed blue light maintainedthe piPSC-colony morphologies for more than 20 weeks, the cells culturedin the dark were fully differentiated after 3 weeks (FIGS. 4B and 4C).The optically maintained alkaline phosphatase-positive piPSCs exhibiteda normal karyotype and comparable expression levels of pluripotencymarkers, such as pOct4, pNanog, pSox2, pLin28a mRNAs and pOct4 protein,to that of the cells maintained with FGF2 supplementation (FIGS. 4D and4E and FIG. 13D-13F). Furthermore, the optically maintained piPSCspossessed the differentiation potential into the three germ layers(FIGS. 4F and 4G), revealing that our optical induction system for FGFsignaling is a suitable culture system for maintaining the pluripotencyof piPSC. Overall, these results support that we successfully developedan efficient and economical FGF2-free PSC culture system.

Simple and cost-efficient LED illumination system. To lower the entrybarriers and increase control of the optical culture system, we nextdeveloped a do it yourself (DIY) light illumination system. Weengineered a multi-well LED illuminating device to optically stimulatestandard 6-well or 12-well tissue culture plates in a conventional CO2incubator (FIG. 5A). The LED illuminating device consists of 6 or 12LEDs of any color, a circuit board, an open-source microcontroller, andan enclosing plastic casing. The LEDs were soldered onto the circuitboard and are controlled by the microcontroller (FIGS. 5A and 5B). Thecasing was designed using computer-aided design software and printedusing a 3D plastic printer (FIG. 5A, 5C). Through our developed softwareand mobile application, the LED illuminating devices are remotelycontrolled by any computer or smartphone via a wireless connection (FIG.5D). Users can easily control the pulse time length, frequency, andintensity of light illumination, and check current status of the LEDillumination system (FIGS. 5E and 5F), which is seamlessly adopted inour routine stem cell culture. This DIY system provides the opportunityto apply optical culture systems to cell cultures in any laboratory.

DISCUSSION

We describe a novel and efficient culture system for PSCs via theactivation of the FGF pathway to maintain PSCs without the need forsupplementation of FGF2 recombinant protein. Previous reports havedemonstrated the optical activation of receptor tyrosine kinases inimmortalized cell lines [31,40,41] and developing embryos [42,43] as aninitial step in evolving such optical technologies. Although a studyusing the optical control of ERK phosphorylation reported a perturbationof patterning and morphogenesis in Drosophila embryos [42], otherstudies did not report any biologically relevant phenotypes; therefore,translating these approaches for use with PSCs has remained challenging.Here, we extend the approach to human and porcine PSCs and provide clearevidence that our Opto-FGFR PSCs are sufficiently maintained by bluelight illumination and without the need for FGF2 proteinsupplementation. Although overall patterns of protein phosphorylation inthe blue light illuminated group were similar to FGF2 protein treatedgroup, MSK1/2, eNOS, and HSP27, which are known to regulate cellproliferation [44-46], were slightly more phosphorylated in the bluelight exposed group than FGF2 treated group (Supplementary FIG. 3 ).Since there is no need to supplement FGF2 protein, the media does notneed to be frequently changed in the optical PSC culture system. Infeeder-free culture systems, very high concentrations of FGF2 (up to 100ng/mL) are required for promoting self-renewal and inhibitingspontaneous differentiation of hPSCs [15]. Even in a feeder-free culturesystem, we succeeded in maintaining pluripotency of hESCs using ouroptical culture system (FIG. 14 ). Moreover, the optically maintainedPSCs appeared to retain the normal differentiation potential oftraditional PSCs.

There are various kinds of photoactivatable proteins available foroptogenetic approaches [9,10]. Using the photolyase homology region ofthe Cry2 protein (Cry2 PHR [47]) and LOV domain [35], two pioneerresearches have established the optical activation systems of the FGFsignaling pathway [31,40]. In the present study, the photosensory LOVdomain served as an initiator in the light-induced intermolecular signaltransduction cascade of the FGF signaling pathway (termed as opto-FGFRsignaling) for maintaining the pluripotency of mammalian PSCs, withoutFGF2 protein supplementation. Although we could optically activate FGFsignaling in human embryonic kidney cells (HEK293T) and hESCstransiently transfected with the Cry2 PHR-based opto-FGFR (FIG. 15 ), weselected the LOV domain as a photodimerizable domain because of tworeasons: the LOV domain has a shorter length (LOV, 432 bp or 144 aa [31]vs Cry2 PHR, 1506 bp or 502 aa [40]), which is more favorable forknock-in, and LOV domain has a shorter dissociation time after lightwithdrawal (LOV t1/2 ¼ 2.5 min. [31] vs Cry2 PHR t1/2 ¼ 5-6 min.[9,10]), which is more advantageous for accurate adjustment of theopto-FGFR signaling in PSC, than others. It is important to select anappropriate photosensory domain as the light-sensing actuator module fora given optogenetic modulation system in PSCs. In future work, it mightalso be important to build a systematic approach to select anappropriate photosensory domain for a given signaling pathway in a givencell type.

The current stem cell culture system is heavily dependent on randomdistribution of expensive and thermo-unstable recombinant proteins in adish, which could jeopardize future mass production of human stem cellsfor a population-wide cell therapy. Our Opto-FGFR PSCs enable opticalactivation without any exogenous FGF2 recombinant proteins and offerspatiotemporally precise optical control of stem cell behaviors. Beyondimplementing a new technology in the stem cell field, our experimentaldata could help lead to the development of a potential therapeutic cureagainst many human diseases by modulating multiple signaling pathwaysother than FGF2 and maximizing the potential of human PSCs. While ouroptogenetic culture system to stimulate the FGF signaling pathway issuitable for maintaining the pluripotency of PSCs, further studies toactivate the LIF and/or Activin signaling via optogenetic stimulationfor generating PSCs in a naïve state of pluripotency [48] will be ofgreat interest. Furthermore, it will be interesting to see whether othersignaling molecules to maintain the pluripotency of PSCs, such asinsulin and/or TGFβ1 (or Nodal) [49], can be replaceable by thephoto-activatable system. Regarding hiPSC culture for potentialtherapeutic application, developing an optogenetic system to activateinsulin and/or TGFβ1 (or Nodal) signaling would be one way to reduce itsproduction costs. In addition, because the FGF signaling pathway is akey factor for several differentiation protocols of PSCs, includingectodermal and neuronal differentiation protocols, our optogeneticsystem could be applied to the differentiation researches.

As many researchers adopted optogenetic techniques particularly forneuroscience field, there is a growing concern of a possiblephototoxicity. For example, other group recently reported aphototoxicity caused by blue light illumination in in vitro culture ofneural cells [50]. However, the light intensity (1 mW/mm²) in theirillumination condition is 1000-fold higher than that of our culturecondition (1 μW/mm²), and the frequency (1 Hz) in their condition is7200-fold higher than that of our condition (0.14 mHz); which are why wedid not detect any noticeable toxicity during over 1-year culture. Itwill be important to know what the susceptible ranges of the lightillumination conditions in different cell types in future is. While itstill needs to study more to find an ideal illumination conditions, ‘5min. blue light illumination in every 2 h’ was sufficient to maintainthe pluripotency of PSCs; further studies to exquisitely optimize theilluminating conditions, such as pulse width, frequency, and intensityof illumination, will be of great interest.

The current animal agriculture and husbandry methods cause significantenvironmental issues such as greenhouse gas emissions, fresh waterconsumption, and arable land usage. As an alternative, cellularagriculture [51] or lab-grown meats [52]′ have been introduced andsocial consent is growing due to ethical and environmentally friendlyconcepts. However, the production costs and use of animal-derivedthermo-unstable recombinant proteins during the cellular agricultureprocess are barriers to entering the market for mass consumption. Basedon our calculation, recombinant FGF2 protein for 1 billion of PSCculture costs $14,000 weekly. In addition, due to thermal andchemo-physical instabilities, FGF2 is recommended to be freshly addedinto the media and it requires additional labor fees. In conclusion, ouropto-FGFR system in livestock PSCs can be a realistic solution todecrease these production costs.

Although researchers may consider that it is too early to know whethergenetic engineering techniques will be safe in the clinic, the resultsof clinical trials of CRISPR/Cas9-mediated genome editing have beenpromising up to now [53-55]. We carefully speculate that theCRISPR-based genome editing of PSCs would be clinically possible withadditional development and troubleshooting. Based on our karyotypicanalysis results, our CRISPR-based genome editing of PSCs did not causedetectable karyotypic abnormalities (FIG. 11F FIG. 13F, and FIG. 14E);however, we will study much details of any possible genetic aberrationsin future. If we employ newly developed genetic engineering techniques,such as prime editors [56], high-fidelity CRISPR/Cas9 nucleases [57],and CRISPR/Cas9 nickase [58], to increase accuracy and to reduceoff-target effects, the safety issues can be mitigated. While it is hardto precisely estimate the cost to produce genetically engineered celllines through the CRISPR system, there would not be an additionalexpense after upfront cost after production.

Currently, there are a few culture optogenetics systems on the market,but these systems are all very expensive. High cost commercial productsprevent large volume optogenetics studies and thus, limit the rate ofresearch in this field. There is also a lack of instrumentation thatallows for precise control of illumination and remote monitoring ofmultiple culture plates. Our DIY illumination system is a hardware,software, and mobile application system capable of precise control andreal-time monitoring of cell culture illumination that can serve as aninexpensive alternative to high cost commercial products. Thisillumination system was designed to be easy-to-assemble, easy-to-use,and low-cost, utilizing 3D printing of thermoplastic materials,programmable open-source electronics, and other readily-availableelectronic parts. Additionally, this system was designed to beadjustable and be customized to the individual researcher. The 3D modelsand source code are publicly available to be edited for any specificneeds. Our cost saving PSC culture system with illumination system couldhelp understanding disease mechanism. Furthermore, we did not find anynotable safety issue, even in the feeder-free condition during theculture period with blue light illumination. Additionally, the LEDilluminating device can be customized with any color LED to be used withany photosynthetic protein of interest. This DIY system provides theopportunity to apply a flexible optical culture system to cell culturesin any laboratory.

CONCLUSION

Our approach for the optical maintenance of human and porcine PSCsprovides new insights for an alternative synthetic approach for the nextgeneration in stem cell research. Furthermore, considering the number ofsignaling pathways that are crucial for controlling cell specificationprocesses, our Opto-FGFR PSCs are a proof-of-concept for the opticalcontrol over a plethora of mammalian stem cell fates.

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1-33. (canceled)
 34. A method for maintaining cells, the method comprising: (a) providing or obtaining, in a culture medium, cells genetically engineered to be maintained by exposure to light; and (b) illuminating the cells with light at a sufficient light intensity and time such that the cells are maintained, thereby maintaining the cells, wherein the method does not require a step of supplementing the culture medium with at least one growth factor required for maintaining the cells.
 35. The method of claim 34, wherein the illuminating of (b) replaces the step of supplementing the culture medium with the at least one growth factor required for maintaining the cells.
 36. The method of claim 34, wherein the cells provided or obtained are capable of self-renewal.
 37. The method of claim 36, wherein, after maintaining the cells, the cells are capable of self-renewal.
 38. The method of claim 34, wherein the cells provided or obtained have the potential to differentiate or to further differentiate into different cell types.
 39. The method of claim 38, wherein, after maintaining the cells, the cells have the potential to differentiate or to further differentiate into different cell types.
 40. The method of claim 34, wherein the cells provided or obtained are stem cells.
 41. The method of claim 40, wherein the stem cells are pluripotent stem cells.
 42. The method of claim 40, wherein the stem cells are embryonic stem cells.
 43. The method of claim 40, wherein the stem cells are induced pluripotent stem cells.
 44. The method of claim 40, wherein the maintaining comprises maintaining pluripotency of the stem cells.
 45. The method of claim 34, wherein the cells provided or obtained are genetically engineered to express a fusion protein comprising a growth factor receptor protein or a portion thereof fused to a photoactivatable domain.
 46. The method of claim 45, wherein the photoactivatable domain is selected from the group consisting of: cryptochrome 2 (Cry2), light, oxygen, and voltage (LOV) domain, phytochrome B (PhyB), and UV-resistance locus 8 (UVR8).
 47. The method of claim 45, wherein the growth factor receptor protein or portion thereof is a fibroblast growth factor receptor (FGFR), a transforming growth factor receptor (TGFR), a portion thereof, or a domain thereof.
 48. The method of claim 47, wherein the growth factor receptor protein or portion thereof is FGFR or an intracellular domain of FGFR.
 49. The method of claim 48, wherein the method results in phosphorylation of ERK1/2.
 50. The method of claim 48, wherein the method does not require a step of supplementing the culture medium with exogenous fibroblast growth factor (FGF).
 51. The method of claim 34, wherein the cells are maintained for at least 1 week.
 52. The method of claim 34, wherein the cells provided or obtained are mammalian cells.
 53. The method of claim 34, wherein the cells provided or obtained are from a livestock animal.
 54. The method of claim 53, wherein the livestock animal is selected from the group consisting of: a pig, a cow, a sheep, and a goat.
 55. The method of claim 34, wherein the cells provided or obtained are from a chicken.
 56. The method of claim 34, wherein the cells provided or obtained are from a human.
 57. The method of claim 34, wherein the light is produced by an illumination source.
 58. The method of claim 57, wherein the illumination source comprises a light-emitting diode (LED).
 59. The method of claim 34, wherein the intensity is from about 0.1 μW/mm² to about 25 μW/mm².
 60. The method of claim 57, wherein the illumination source: (i) illuminates the cells for a period of time from about 1 minute to about 120 minutes; (ii) illuminates the cells in a time interval of from about 30 minutes to about 4 hours; or (iii) both (i) and (ii).
 61. The method of claim 57, wherein the illumination source continuously illuminates the cells.
 62. The method of claim 57, wherein the illumination source illuminates the cells with a pulsed light pattern.
 63. The method of claim 57, wherein the illumination source spatiotemporally controls illumination of the cells. 